[Expand] Transformation of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Transformation of Phytochrome B for protein fusion.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator
Investigator: Jeff, Rosario
Aim of the experiment: Miniprep of pTUM100 with pGAL, pTEF1, pTEF2, pADH and RFC25 compatible RFP generator
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Sequencing of RFP-Generator (RFC25, pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Sequencing of RFP-Generator (RFC25, pSB1C3)
Procedure:
Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer)
Tuesday, April 23rd
[Expand] Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Picking of of E. coli XL1 blue with Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Procedure:
pSB1C3 plasmid with BBa_K801031 (PhyB 2 - 908 aa, RFC25): Colonies were picked from chloramphenicol plates.
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
4 colonies were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight
[Expand] Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5)
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of RFP-generator (RFC25, pSB1C3, P4 & P5).
Procedure:
Batch for analytical digestion for P4 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P4
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Batch for analytical digestion for P5 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P5
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
P4
P5
Mutation successful
Mutation successful!
Parts are compliant and do not contain RFC25 forbidden restriction sites.
[Expand] Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2
Investigator: Jeff, Rosario, Florian
Aim of the experiment: Sequencing of pTUM vectors with pGAL, pADH, pTEF1, pTEF2
Procedure:
Sequencing batch were prepared after manufacturer's protocol. (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different vectors we sequenced received the following barcodes:
- ADH in pTUM100: FR01002265
- TEF1 in pTUM100: FR01002266
- TEF2 in pTUM100: FR01002266
- GAL in pTUM100: FR01002268
Sequencing of TEF2 in pTUM100 was not interpretable. The other sequences were consistent with the sequences in the parts registry.
Wednesday, April 24th
[Expand] Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3)
Investigator: Jeff, Florian
Aim of the experiment: Miniprep of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10
Investigator: Jeff, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of Phytochrome B (2-908 N-terminal amino acids) (BBa_K801031, RFC25, pSB1C3), P7 - P10.
Procedure:
Batch for analytical digestion for P7 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P7
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Batch for analytical digestion for P8 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P8
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Batch for analytical digestion for P9 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P9
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Batch for analytical digestion for P10 with NgoMIV+AgeI-HF
volume
reagent
2.5 µl
Plasmid DNA P10
2 µl
NEBuffer 4 (10x)
0.25 µl
NgoMIV (10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
P7
P8
P9
P10
Part is correct
Part is correct
Part is correct
Part is correct
[Expand] Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)
Investigator: Jeff, Florian
Aim of the experiment: Transformation of E. coli XL1 blue with EreA and EreB (pDEST14).
Procedure:
Plasmid DNA was received dried in paper from McMaster University.
DNA was resuspended in ddH2O
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.
Week 2
Monday, April 29th
[Expand] Miniprep of pRK792, pRK793, pRIL, ereA, ereB
Investigator: Rosario
Aim of the experiment: Miniprep of pRK792, pRK793, pRIL, ereA, ereB
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Concentrations were determined by measuring the absorption:
Plasmid
c [ng/µl]
pRK792
214,5
pRK793
154,3
pRIL
6,1
ereA
183,4
ereB
98,3
The procedure will have to be repeated for pRIL
[Expand] Midiprep of RFC25 compatible RFP generator in (pSB1C3)
Investigator: Andreas, Florian
Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)
Procedure:
Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)
Tuesday, April 30th
[Expand] Preparative digestion and gelelectrophoresis of mINF1 and Leptin with HindIII and EheI
Investigator: Louise, Johanna
Aim of the experiment: Testing the enzymes, since the earlier digestion with probably older enzymes did not work (no bands on gel) .
Procedure:
Batch for preparative digestion for mINF1 with HindIII and EheI
volume
reagent
10 µl
Plasmid DNA mINF11
5 µl
Tango Buffer (10x)
2 µl
HindIII (10 U/µl)
3 µl
EheI (10 U/µl)
30 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion for Leptin with HindIII and EheI
volume
reagent
20 µl
Plasmid DNA Leptin
5 µl
Tango Buffer (10x)
2 µl
HindIII (10 U/µl)
3 µl
EheI (10 U/µl)
20 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
[Expand] Transformation of E. coli XL1 blue with PSH21 (P17)
Investigator: Johanna, Louise
Aim of the experiment: Transformation of E. coli XL1 blue with PSH21.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.
5 µl of DNA was added to 250 µl of competent cells and gently mixed.
60 min incubation on ice
5 min. heat shock at 37 °C
Adding the DNA to 2 ml LB-medium.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.
Thursday, May 2nd
[Expand] Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system
Investigator: Katrin
Aim of the experiment: Miniprep of pSH21 (pAutoRex8) containing AlcR/AlcA promotor system
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The concentration was measured (900 ng/µl)
Friday, May 3rd
[Expand] Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry
Investigator: Andreas
Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.
5 µl of DNA was added to 250 µl of competent cells and gently mixed.
60 min incubation on ice
5 min. heat shock at 37 °C
Adding the DNA to 2 ml LB-medium.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.
[Expand] Plating of received E. coli containing biobricks
Investigator: Jeff, Andreas
Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.
Operational sequence:
Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.
There were no colonies on the plate with K864401 (p33), therefore the LB medium could not be infected
The tubes were placed into the incubator at 37 °C
Week 3
Monday, May 6th
[Expand] Miniprep of the prepared E. coli containing biobricks and the parts from LMU
Investigators: Florian, Johanna, Jefferey
Aim of the experiment: A miniprep of the overnight culture of our E. coli containig our biobricks and LMU parts was performed using the Qiagen miniprep kit.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
Resulting concentrations were determined by measuring the absorption:
Label
Name
Concentration [ng/µl]
p19
mCherry in pSB1C3
40,2
p20
GFP in pSB1C3
58,5
p21
mKate2 in pSB1C3
131,3
p22
35S Promoter + (Strong Plant RBS + GFP) From K414001
42,8
p23
constitutive promoter CaMV35S
39,3
p24
cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
30,0
p25
mStrawberry
46,6
p26
DREB1C promoter
34,8
p27
bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode
p45
FluA
FR01002355
p28
SV40
FR01002356
p23
CaMV35S
FR01002357
p38
xylE
FR01002358
p21
LMU mKate2
FR01002359
p20
LMU GFP
FR01002360
p19
LMU mCherry
FR01002345
p46
mCherry
FR01002346
p26
DREB1C
FR01002347
p35
RD29A
FR01002348
[Expand] Preparation of ordered primers
Investigators: Andreas, Jefferey, Rosario
Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl
Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.
Primers were labeled as follows:
Label
Oligonr.
6
EreA_for
7
EreA_rev
8
EreA_SDM1_for
9
EreA_SDM1_rev
10
EreA_SDM2_for
11
EreA_SDM2_rev
12
EreA_SDM3_for
13
EreA_SDM3_rev
14
EreB_for
15
EreB_rev
16
EreB_SDM1_for
17
EreB_SDM1_rev
18
EreB_SDM2_for
19
EreB_SDM2_rev
20
EreB_SDM3_for
21
EreB_SDM3_rev
Tuesday, May 7th
[Expand] PCR, purification and analytical geleletrophoresis of EreA (P15) and EreB (P16)
Investigator: Jeff, Louise, Florian, Rosario, Andi, Johanna
Aim of the experiment: PCR of EreA (P15) and EreB (P16).
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P15: O46 (EreA_for); P16: O31 (EreB_for))
1 µl
10 µM Reverse Primer (P15: O47 (EreA_rev); P16: O32 (EreB_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P15; P16)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The gradient PCR program was performed after following scheme with following conditions (Tm=54 °C; ΔG=2 °C; P15 in row 11(=52 °C); P16 in row 1 (=54.0 °C)):
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
Tm=54 °C; ΔG=2 °C
60 s
68 °C
80 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
9 µl of the purified PCR products were mixed with 1 µl DNA loading buffer (10x) for analytical gelelectrophoresis
Analytical gelelectrophoresis was performed at 90 V for 60 min in 1% agarose gel.
1 kbp ladder
F1
F2
Fragment-size like expected
Fragment-size like expected
[Expand] Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV
Investigator: Jeff, Andi, Florian, Rosario, Louise, Johanna
Aim of the experiment: Preparative digestion and gelelectrophoresis of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI as well as 20aalinker-PIF3 (P51) with EcoRI and NgoMIV .
Procedure:
Batch for preparative digestion of PhyB (P9) and PIF3-20aa linker (P50) with AgeI and PstI
volume
reagent
20 µl
Plasmid DNA P9/P50
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
PstI-HF
13.6 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of 20aalinker-PIF3 (P51) with EcoRI and NgoMIV
volume
reagent
20 µl
Plasmid DNA P51
4 µl
NEBuffer 4
1 µl
EcoRI-HF
1 µl
NgoMIV
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 3 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the reaction batches after digestion and were loaded on an 1% low-melting agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 70 V for 90 min.
P9 digestion = F3
1 kbp ladder
P50 digestion = F4
P51 digestion = F5
Fragment-size like expected; band was cut out
Fragment-size like expected; band was cut out
Fragment-size like expected; band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Wednesday, May 8th
[Expand] Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI
Investigator: Jeff, Andi, Florian, Rosario, Louise
Aim of the experiment: Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.
Procedure:
Batch for preparative digestion of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.
volume
reagent
25 µl
PCR product F1/F2
5 µl
NEBuffer 4
0.5 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
17.5 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P52 for preperation of pSB1C3 vector:
volume
reagent
20 µl
Plasmid DNA P52
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
13.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the reaction batches for F1 & F2 and 4 µl of DNA loading buffer (10x) were added to the reaction batches for Pxx after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 60 min.
F1 digestion = F6
F2 digestion = F7
1 kbp ladder
P52 digestion = F8
Length was like expected
Length was like expected
Length was like expected; the lower band was extracted
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3)
Investigator: Jeff, Andi, Florian, Rosario, Louise
Aim of the experiment: Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3). Quickchanges have still to be performed.
Procedure:
Ligation batch for F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3):
Ligation batch for F8+F6
volume
reagent
1.35 µl
F8 (74.3 ng/µl, 2086 bp)
6.76 µl
F6 (25.9 ng/µl, 1218 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
8.89 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F8+F7
volume
reagent
1.35 µl
F8 (74.3 ng/µl, 2086 bp)
8.78 µl
F7 (20.6 ng/µl, 1257 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
6.87 µl
ddH2O
=20 µl
TOTAL
Negative control was als prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batches.
Cycled ligation has been performed after following protocol in a thermocycler:
100 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
[Expand] Midiprep of cloning vector RFC25 RFP generating device
Investigators: Andreas, Rosario
Aim of the experiment: Midiprep of our cloning vector RFC25 RFP generating device.
Procedure: Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)
The yield of our vector was 1854 ng.
Thursday, May 9th
[Expand] Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB)
Aim of the experiment: Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB). Quickchanges has now to be performed to remove forbidden restriction sites.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of ligation products F8+F6, F8+F7, F8 negative control were added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Friday, May 10th
[Expand] Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs
Investigator: Andi, Jeff, Rosario
Aim of the experiment: Deletion of found mutations.
Operational sequence
1. PCR general setup
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
DNA template
1 µl
1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
DNA template
1 µl
1:10 dilution of appropriate Oligo (= 5 pmol)
16.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
12
95 °C
30 s
55 °C
1 min
68 °C
6 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
After the PCR was finished 1 µl of the DpnI restriction enzyme (10 U/µl)was added
Now the reaction was mixed gently and thoroughly, spinned down in a microcentrifuge for 1 minute, and immediately incubated at 37 °C for 1 hour to digest the parental supercoiled dsDNA
2. Used constructs and primers
Quickchange number
Plasmid number
Geneconstruct
Oligo number
QC 1
P27
BPul_Laccase+ pSB1C3
O26 & O27
[Expand] Picking of transformed Plasmids F8 + F6
Investigator: Andi
Aim of the study: Picking from the overnight transformation of the ligated F8+F6, to see whether ligation is successful or not.
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
[Expand] PCR of TEV (P14, pRK793)
Investigator: Katrin, Jeff
Aim of the experiment: PCR, purification and analytical geleletrophoresis of TEV (P14, pRK793) to produce TEV with RFC25 pre- and suffx (still contains forbidden SpeI-site).
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (O34 (TEV_fw))
1 µl
10 µM Reverse Primer (O35 (TEV_rv))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P14)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme with following conditions:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
55 °C
60 s
68 °C
150 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Resulting product was labeled as F12.
[Expand] Digestion of P61 with S1 Nuclease
Investigator: Katrin
[Expand] Oligohybridization of O22 (MinMCS_for) and O23 (MinMCS_rev)
Investigator: Andi
Aim of the experiment: Oligohybridization of MinMCS_for (O22) and MinMCS_rev (O23
Procedure:
25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev
Heating up to 95 °C for 5 min
Cooling at RT in a styropor box overnight
[Expand] Preparation of ordered primers
Investigators: Andreas, Rosario, Katrin, Ingmar
Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl
Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.
Primers were labeled as follows:
Label
Oligonr.
24
BPU_for
25
BPU_rev
26
BPU_SDM1_for
27
BPU_SDM1_rev
28
BPU_SDM2_for
29
BPU_SDM2_rev
30
BPU_SDM3_for
31
BPU_SDM3_rev
32
BPU_SDM4_for
33
BPU_SDM4_rev
34
TEV_for
35
TEV_rev
36
TEV_t387c_for
37
TEV_t387c_rev
38
N_Split_TEV_for
39
N_Split_TEV_rev
40
C_Split_TEV_for
41
C_Split_TEV_rev
42
PIF6_2-100_for
43
PIF6_2-100_rev
44
p104AgeI a71g fw
45
p104AgeI a71g rev
46
xylE_for
47
xylE_rev
48
xylE_c312a_for
49
xylE_c312a_rev
50
xylE_g483a_for
51
xylE_g483a_rev
52
xylE_a834g_for
53
xylE_a834g_rev
Saturday, May 11th
[Expand] Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI
Investigator: Louise
Aim of the experiment: Preparative digestion of PCR product of S219V pRK793 (F12) with XbaI & AgeI. Quickchange has still to be performed (forbidden SpeI restriction site).
Procedure:
Batch for preparative digestion of PCR product S219V pRK793 (F12) with XbaI & AgeI.
volume
reagent
20 µl
PCR product F12
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
13.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 3 h at 37 °C
Digested products were purified to remove nucleotides and enzymes with QIAquick PCR Purification Kit, QIAGEN.
The digested fragment was labelled F15
[Expand] Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA)
Investigator: Louise
Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA) of 2 clones picked the day before
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The plasmids were labelled as P63 and P64
[Expand] Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI
Investigator: Louise
Aim of the experiment: Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI
Procedure:
Batch for analytical digestion of P63 and P64 with AgeI-HF and XbaI
volume
reagent
2.5 µl
Plasmid DNA P63/P64
2 µl
NEBuffer 4 (10x)
0.2 µl
BSA (100x)
0.25 µl
XbaI(10 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
14.8 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Analytical digestion of P63 with AgeI-HF and XbaI
1 kbp ladder
Analytical digestion of P64 with AgeI-HF and XbaI
Insertion successful
Insertion successful
[Expand] Preparative digestion of PCR product of EreB (F2) with XbaI & AgeI
Investigator: Louise, Johanna
Aim of the experiment: Preparative digestion of PCR product EreB (F2) with XbaI & AgeI
(second try since there did not grow any transformed bacteria in the first time).
Procedure:
Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume
reagent
20 µl
PCR product F2
5 µl
NEBuffer 4
0.5 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
22.5 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C
The digested fragment was labelled F14
[Expand] Purification and ligation of digested PCR product EreB (F14)
Investigator: Louise, Rosario
Aim of the experiment: PCR Purification and ligation of digested PCR product EreB (F14).
Procedure:
PCR Purification of EreB PCR (F14)
To purify the digested Fragment(F14) the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be 5 ng/µl via Nano drop.
Ligation of F14 + F8(pSB1C3)
Ligation batch for F8+F14 (positive control)
volume
reagent
1.35 µl
F8 (74.3 ng/µl, 2086 bp)
15.65 µl
F14 (5 ng/µl, 1257 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
[Expand] Transformation of ligation products of F10 + F11 into E.coli Xl1-Blue
Investigator: Andi
Aim of the experiment:Transformation of the ligation products of F10 + F11 and the negative control in pTUM104 in XL1 Blue.
Operation Sequence
melting of 2x 200 µl Ca-competent E.coli XL1-Blue cells on ice
Preparing 4 Tubes with 100 µl of Ca-competent E.coli XL1-Blue cells
addition of 5 µl of the ligation products
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate
[Expand] Transformation of Quickchange Product P62 into E.coli Xl1-Blue
Investigator: Andi
Aim of the experiment:Transformation of the Quickchange Product P62 and the negative control in XL1 Blue.
Operation Sequence
melting of 1x 200 µl Ca-competent E.coli XL1-Blue cells on ice
Preparing 4 Tubes with 50 µl of Ca-competent E.coli XL1-Blue cells
addition of 1 µl of the Quickchange Product
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate
[Expand] Quickchange mutagenesis of pTUM104
Investigator: Ingmar
Aim of the experiment: Sequencing results showed a forbiden AgeI restriction site in this vector. The sdm will delete this restricition site
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P6 template
0.5 µl
1:10 dilution of O44 (10 pmol/µL)
16.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P6 template
0.5 µl
1:10 dilution of O45 (10 pmol/µL)
16.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
10
95°C
30 sec
55°C
1 min
67°C
6 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C. The resulting product was named fragmet 14.
Transformation into E.coli Xl1-BlueOperation Sequence
melting of 100 µl Ca-competent E.coli XL1-Blue cells on ice
addition of 1 µl of the PCR product
incubation for 30 min on ice
heat shock for 5 min at 37 °C
transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
plate 100 µl on an Amp-LB-plate
sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate
Sunday, May 12th
[Expand] Analytical gelelectrophoresis of F15 (digested TEV PCR product)
Investigator: Jeff, Rosario
Aim of the experiment: Analytical gelelectrophoresis of F15 (digested TEV PCR product) to check whether PCR has worked.
Procedure:
5 µl of F15 was mixed with 0.55 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.
PCR product has expected length
[Expand] Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25)
Investigator: Jeff, Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F15 (TEV in pSB1C3, RFC25). Further quickchanges still have to be done.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Transformation of E. coli XL1 blue with ligation products F8+F14 (EreB in pSB1C3, RFC25)
Investigator: Jeff, Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F14 (EreB in pSB1C3, RFC25). Further quickchanges still have to be done.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Transformation of E. coli XL1 blue with P23
Investigator: Jeff, Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with P23 for insertion of miniMCS.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Picking of transformed Plasmid F10+F11 (IRES from polio virus, blunt ligated with pSB1C3 for sequencing)
Investigator: Andi
Aim of the study: Picking of transformed Plasmid F10+F11 (AlcR blunt ligated with pSB1C3 for sequencing).
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
10 colonies were picked.
Week 4
Monday, May 13th
[Expand] Miniprep of overnight culture of transformated E.~coli XL1-Blue with F10+F11
Investigator: Rosario, Jeffery, Johanna, Flo
Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue F10+F11 of 10 clones picked the day before
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analyzing sequencing results
Investigator: Johanna
Aim of the experiment: Comparison of the sequencing results with the Biobrick (BB) sequences.
Procedure: Using Geneious to align the sequencing results with the BB sequences.
Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 65.
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid P27 template
0.5 µl
1:10 dilution of O26 (10 pmol/µL)
20. 5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid P27 template
0.5 µl
1:10 dilution of O27 (10 pmol/µL)
20.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
10
95°C
30 sec
55°C
1 min
67°C
4 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles was increased to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Quickchange mutagenesis SDMI of p38
Investigator: Jeff, Flo, Andi, Rosario
Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 67.
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0,5 µl
Plasmid P38 template
0.5 µl
1:10 dilution of O48 (10 pmol/µL)
20.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid P38 template
0.5 µl
1:10 dilution of O49 (10 pmol/µL)
20.5 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
10
95°C
30 sec
55°C
1 min
67°C
4 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Quickchange mutagenesis SDMI of P63
Investigator: Jeff, Rosario, Flo, Andi
Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled P66.
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0.25 µl
Plasmid P63 template; 1:2 dilution
0.5 µl
1:10 dilution of O8 (10 pmol/µL)
20.75 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
0.25 µl
Plasmid P63 template; 1:2 dilution
0.5 µl
1:10 dilution of O9 (10 pmol/µL)
20.75 µl
ddH2O
0.5 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
10
95°C
30 sec
55°C
1 min
67°C
6 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, the amount of cycles was raised to 20
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Transformation of E. coli XL1 blue with P65
Investigator: Jeff, Rosario, Johanna, Andi, Flo
Aim of the experiment: Transformation of E. coli XL1 blue with P65 .
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Transformation of E. coli XL1 blue with P66
Investigator: Jeff, Rosario, Flo, Johanna, Andi
Aim of the experiment: Transformation of E. coli XL1 blue with P66.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Transformation of E. coli XL1 blue with P67
Investigator: Jeff, Rosario, Johanna, Andi, Flo
Aim of the experiment: Transformation of E. coli XL1 blue with P67.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Tuesday, May 14th
[Expand] Analytical digestion of P15 and P19 to P52
Investigator: Florian, Johanna
Aim of the experiment: Analytical digestion and gelelectrophoresis of P15 and P19 to P52 to check the sequencing results.
Procedure:
Batch for analytical digestion of P15 and P19 to P52
volume
reagent
5 µl
Plasmid DNA
5 µl
Mastermix
=10 µl
TOTAL
Batch of Mastermix
volume
reagent
40 µl
NEBuffer 4 (10x)
4 µl
BSA (100x)
0.2 µl
NotI(10 U/µl)
148 µl
ddH2O
=200 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Discussion:
Gel 1, Row 1:
lane
part
plasmid
band 1
band 2
result
2
EreA
pDEST14
3,25 kb
no cut, no restriction sites in pDEST14
3
mCherry(LMU)
pSB1C3
2,0 kb
750 bp
probably right
4
GFP(LMU)(845 bp)
pSB1C3
2,0 kb
750 bp
maybe switched with mKate2(LMU)
5
mKate2(LMU)(702 bp)
pSB1C3
2,0 kb
900 bp
maybe switched with GFP(LMU)
6
35S + Strong Plant + GFP(1775 bp)
pSB1C3
2,0 kb
750 bp
?
7
UVR8(1332 bp)
pSB1C3
2,0 kb
1,1 kb
?
8
mStrawberry(708 bp)
pSB1C3
2,0 kb
1,4 kb
?
9
DREB1C promoter(463 bp)
pSB1C3
2,5 kb
probably only one cut -> one NotI site mutated
10
Laccase(1587 bp)
pSB1C3
2,0 kb
1,3 kb
?
11
SV40(419 bp)
pSB1C3
2,0 kb
1,6 kb
this could be the laccase
12
cjBlue(696 bp)
pSB1C3
2,0 kb
0,5 kb
?
Gel 1, Row 2:
lane
part
plasmid
band 1
band 2
result
2
alcA (843 bp)
pSB1C3
2,0 kb
0,7 kb
?
3
eforRed (681 bp)
pSB1C3
plasmid
plasmid bands (coiled, supercoiled, etc.)
4
amilGFP (699 bp)
pSB1C3
2,0 kb
0,7 kb
right part
5
mCFP (714 bp)
pSB1C3
2,0 kb
0,7 kb
right part
6
RD29A promoter (1535 bp)
pSB1C3
2,0 kb
1,5 kb
right part
7
amilCP (669 bp)
pSB1C3
2,0 kb
0,7 kb
right part
8
CMV promoter (580 bp)
pSB1C3
2,0 kb
0,6 kb
right part
9
middle linker (24 bp)
pMA-BBFR
2,4 kb
short insert went through the gel
10
GSAT (108 bp)
pMA-BBFR
2,4 kb
0,1 kb
right part
11
GFP(720 bp)
pSB1A2
?
12
long linker (36 bp)
pMA-BBFR
2,4 kb
short insert went through the gel
Gel 2:
lane
part
plasmid
band 1
band 2
result
2
SEG linker (108 bp)
pMA-BBFR
2,4 kb
short insert went through the gel
3
short linker (12 bp)
pMA-BBFR
2,4 kb
short insert went through the gel
4
FluA (522 bp)
pMA-BBFR
2,4 kb
0,5 kb
right part
5
mCherry (769 bp)
pSB2K3
4,4 kb
0,75 kb
right part
6
Cerulean (778 bp)
pSB2K3
?
7
mRFP1 (706 bp)
pSB2K3
4,4 kb
0,7 kb
right part
8
mOrange (769 bp)
pSB2K3
4,4 kb
0,75 kb
right part
9
PIF3-20aa (366 bp)
pSB1C3
2,0 kb
0,35 kb
right part
10
20aa-PIF3 (366 bp)
pSB1C3
2,0 kb
0,35 kb
two plasmids and two insert can be seen
11
LexA (ca. 6 kb)
pSB1C3
> 8 kb
only one cut, huge plasmid
[Expand] Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV)
Investigator: Jeff, Florian
Aim of the study: Picking of Plasmid P23 (CaMV35S) and Ligation Product F8 + F15 (TEV) for Miniprep.
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
1 colony of P23 were picked.
7 colonies of F8 + F15 were picked.
[Expand] Sequencing of P63 & P64
Investigators: Louise
Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
p63
EreA
FR01002349
FR01002350
p64
EreA
FR01002351
FR01002352
Wednesday, May 15th
[Expand] Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI
Investigator: Louise,Johanna
Aim of the experiment:Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI to recover EreA and prepare two new vectors for cloning.
Procedure:
Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume
reagent
20 µl
Plasmid (P8,P60,P64)
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
13.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C
The digested fragments were labelled F17(P8), F18(P60), F19(P64)
[Expand] PCR, purification and analytical geleletrophoresis of and EreB (P16)
Investigator: Jeff, Louise
Aim of the experiment: PCR of EreB (P16).
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (O31 (EreB_for))
1 µl
10 µM Reverse Primer (O32 (EreB_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P15; P16)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program TMKS was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
52 °C
60 s
68 °C
80 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
4 µl of the purified PCR products were mixed with 0.4 µl DNA loading buffer (10x) for analytical gelelectrophoresis
Analytical gelelectrophoresis was performed at 90 V for 30 min in 1% agarose gel.
1 kbp ladder
F16
Fragment-size like expected
[Expand] Purification and ligation of digested products F17, F18, F19
Investigator: Jeff, Louise
Aim of the experiment: Purification and ligation of digested products F17, F18, F19.
Procedure:
To purify the digested Fragments the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be XX ng/µl via Nano drop.
Ligation of F19 + FXX(pSB1C3)
Ligation batch for F19+FXX (positive control)
volume
reagent
1.35 µl
F8 (74.3 ng/µl, 2086 bp)
X µl
F14 (5 ng/µl, 1257 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
[Expand] Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI)
Investigator: Jeff
Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P90-P97 (TEV protease, XbaI+AgeI) and P97 (CaMV p35S, XbaI+PstI).
Procedure:
Mastermix for analytical digestion of P90-P96 with XbaI+AgeI-HF:
volume
reagent
16 µl
NEBuffer 4 (10x)
1.6 µl
BSA (100x)
2 µl
XbaI (20 U/µl)
2 µl
AgeI-HF (20 U/µl)
118.4 µl
ddH2O
=140 µl
TOTAL
Batch for digestion of P90-P96 with XbaI+AgeI-HF:
volume
reagent
2.5 µl
Plasmid DNA
17.5 µl
Mastermix
=20 µl
TOTAL
Batch for digestion of P90-P96 with XbaI+AgeI-HF:
volume
reagent
2.5 µl
Plasmid DNA
2 µl
NEBuffer 4 (10x)
0.2 µl
BSA (100x)
0.25 µl
XbaI (20 U/µl)
0.25 µl
AgeI-HF (20 U/µl)
14.8 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
Digestion of P90
Digestion of P91
Digestion of P92
Digestion of P93
Digestion of P94
Digestion of P95
Digestion of P96
Digestion of P97
Insert has expected length
Insert has expected length
Insert has expected length
Insert has expected length
Corrupt
Insert has expected length
Insert has expected length
Corrupt
The inserts for TEV semm to be right in the most cases, but the backbone has mutated and it should be cloned into a new pSB1C3 with new same restriction enzymes (XbaI+AgeI)!
The forward sequencing of CaMV p35S is right but the analytical digestion is wrong. Maybe further parts are downstream of the promoter?! Reverse sequencing should be performed for further analysis.
Thursday, May 16th
[Expand] Preparative digestion of pSH21 (P61), psB1A2(P41) and P96 (TEV S19V)
Investigator: Jeff, Katrin
Aim of the experiment: Preparative digestion of pSH21 (P61)and psB1A2(P41) with EcoRI and P96 (TEV S19V) with XbaI & AgeI for subsequent ligation.
Procedure:
Batch for preparative digestion of pSH21 with EcoRI
volume
reagent
20 µl
Plasmid DNA P61
4 µl
Buffer 4(10x)
1 µl
EcoRI (20 U/µl)
15 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of psB1A2 with EcoRI
volume
reagent
20 µl
Plasmid DNA P41
4 µl
Buffer 4(10x)
1 µl
EcoRI (20 U/µl)
15 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P96 (TEV S219V) with XbaI & AgeI.
volume
reagent
20 µl
Plasmid DNA P96
4 µl
Buffer 4(10x)
1 µl
XbaI (20 U/µl)
1 µl
AgeI-HF (20 U/µl)
13.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 3 h at 37 °C.
The resulting fragments were named: F23 (P41) and F22 (P61)
4 µl of DNA loading buffer (10x) were added to the reaction batch containing pSH21 (P61) after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 70 V for 90 min.
1 kbp ladder
P61 digestion = F23
Fragment-size like expected; band was cut out
The band had the expected length. They were cut out and purified using the QIAquick Gel Extraction Kit, QIAGEN
[Expand] Preparative digestion of ereB (P64) with XbaI & AgeI.
Investigator: Jeff, Louise, Katrin
Aim of the experiment:Preparative digestion of EreB (P64) with XbaI & AgeI.
Procedure:
Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.
volume
reagent
20 µl
Plasmid
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
13.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C
The digested fragments were labelled
[Expand] Dephosphorylation of F23
Investigator: Jeff, Katrin
Aim of the experiment: Dephosphorylation of cut vector psB1A2 to prevent re-ligation
Procedure:
the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer
Batch for the dephosphorylation of F23
volume
reagent
30 µl
Plasmid DNA F23 (2 µg)
4 µl
10X Fast AP buffer
2 µl
FastAP Thermosensitive Alkaline
Phosphatase
4 µl
ddH2O
=40 µl
TOTAL
the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
the reaction was stopped by heating to 75 °C for 5 minutes
[Expand] Ligation of pSH21 (F22) with psB1A2(F23), TEV (P96), EreA (P64), pSB1A2(P41)
Investigator: Jeff, Katrin
Aim of the experiment: ligation of pSH21 (F22) with pSB1A2(F23), TEV (F20) with pSB1C3 (F17), EreA (F19)with pSB1C3 (F17) and EreB (F21) with pSB1C3 (F17)
Procedure:
Ligation batch for EreB (F21) with pSB1C3 (F17)
volume
reagent
1.6 µl
F17 (61,4 ng/µl)
15,4 µl
F21 (8,3 ng/µl)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for TEV (F20) with pSB1C3 (F17)
volume
reagent
1.6 µl
F17 (61,4 ng/µl)
4,1 µl
F20 (24,7 ng/µl)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
11,3 µl
ddH20
=20 µl
TOTAL
Ligation batch for pSH21 (F22) with pSB1A2(F23)
volume
reagent
0,85 µl
F23 (118,1 ng/µl)
2,3 µl
F22 (119,0 ng/µl)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
13,85 µl
ddH20
=20 µl
TOTAL
Ligation batch for EreA (F19) with pSB1C3 (F17)
volume
reagent
1.6 µl
F17 (61,4 ng/µl)
6,4 µl
F19 (8,3 ng/µl)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
9 µl
ddH20
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
the ligation was performed at 16 °C overnight
Friday, May 17th
[Expand] Transformation of E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23, F17 (negative control), F23 (negative control)
Investigator: Jeff, Katrin
Aim of the experiment: Transformation of ligation products F17+F21 (pSB1C3, EreB), F17+F20 (pSB1C3, TEV), F17+F19 (pSB1C3, EreA), F23+F22 (pSB1A2, pSH21) and negative controls
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on chloramphenicol plates (F17+F19, F17+F20, F17+F21, F17 negative) or ampicillin plates (F23+F22, F23 negative control)
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (F17+F19, F17+F20, F17+F21) or ampicillin plates (F23+F22)
[Expand] Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs
Investigator: Jeff
Aim of the experiment: removal of forbidden restiction sites from laccase
[Expand] Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33
Investigator: Jeff, Andi, Johanna
Aim of the experiment: Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on chloramphenicol plates 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33)
Saturday, May 18th
[Expand] Picking of transformed Plasmid E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23
Investigator: Andi, Jeff
Aim of the study: Picking of transformed Plasmid F17+F19, F17+F20, F17+F21, F22+F23 .
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR/AmpR LB-medium)
2 colonies were picked for every Transformation product.
Sunday, May 19th
[Expand] Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23
Investigator: Jeff, Andi
Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with F17+F19, F17+F20, F17+F21, F22+F23 of 2 clones picked the day before
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and analytical gelelectrophoresis of F17+F19, F17+F20, F17+F21 and F22+F23
Investigator: Jeff, Andi
Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of ligation products F17+F19, F17+F20, F17+F21, F22+F23.
Procedure:
Mastermix for analytical digestion of F17+F19, F17+F20, F17+F21 with XbaI+PstI-HF:
volume
reagent
14 µl
NEBuffer 4 (10x)
1.4 µl
BSA (100x)
1.75 µl
XbaI (20 U/µl)
1.75 µl
AgeI-HF (20 U/µl)
103.6 µl
ddH2O
=122.5 µl
TOTAL
Batch for digestion of F22+F23 with EcoRI-HF:
volume
reagent
2.5 µl
Plasmid DNA
2 µl
NEBuffer 4 (10x)
15.25 µl
ddH2O
0.25 µl
EcoRI-HF (20 U/µl)
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
Digestion of F17+F19
Digestion of F17+F19
Digestion of F17+F20
Digestion of F17+F20
Digestion of F17+F21
Digestion of F17+F21
Digestion of F22+F23
Digestion of F22+F23
Insert has expected length
Insert has expected length, the first line represents the rest of the uncut Plasmid
Insert has expected length
Insert has expected length, the first line represents the rest of the uncut Plasmid
Insert has expected length
Insert has expected length
Insert has expected length, insert and cut Plasmid overlay each other, because of the same length
Corrupt
[Expand] Quick Change mutagenesis to remove forbidden restriction sites of P28 (Laccase), P100 (TEV S219V), P102 (EreB)
Investigator: Jeff, Andi
Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful
Investigator:
Aim of the experiment:
Procedure:
[Expand] Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue
Investigator: Jeff, Andi
Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Week 5
Tuesday, May 21st
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMI - of P28 (Laccase), P100 (TEV S219V), P102 (EreB)
Investigator: Jeff, Andi, Johanna
Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P100 (TEV S219V), P102 (EreB).
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Analytical gelelectrophoresis of QuickChange products of P100, P102 and P28 to check whether the QuickChange PCR and DpnI digestion were successful
Investigator:
Aim of the experiment:
Procedure:
[Expand] Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue
Investigator: Jeff, Andi, Johanna
Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Sequencing of P105, P28, P100, P102 & P98
Investigators: Jeff, Andi,Johanna
Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
p105
IRES
FR01002288
FR01002269
p102
EreB
FR01002270
FR01002275
p100
TEV S219V
FR01002278
FR01002279
p98
EreA
FR01002276
FR01002277
p28
Laccase
FR01002280
[Expand]Preparation of Knop-Medium for Moss
Investigator: Jeff, Andi, Johanna, Rosario
Aim of the experiment: Preparation of Knop-Medium for Moss
Procedure:
All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
Volume of prepared Medium: 1L
10 ml of each stock were converted into a 1,8L Erlenmeyer flask and filled up to 1L with destilled water (ELGA); 12,5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH
The prepared Volume of 1L was shared equally to 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium
All flasks have been autoclaved
[Expand] Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)
Investigator: Andi, Jeff, Johanna, Rosario
Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17) from Sunday, May 19th.
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
3 colonies for P100QC (TEV, QC with O36/O37) and 5 colonies from P102QC (EreB, QC with O16/O17) were picked from the plates.
Wednesday, May 22nd
[Expand] Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17)
Investigator: Jeff
Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with QuickChange products of P100 (TEV, QC with O36/O37) and P102 (EreB, QC with O16/O17).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27)
Investigator: Jeff
Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P28 (Laccase, QC with O26/O27).
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
8 colonies for P28QC (Laccase, QC with O26/O27) were picked from the plates.
[Expand] Analytical digestion and analytical gelelectrophoresis of P106, P107, P108, P109, P110, P111, P112, P113
Investigator: Jeff, Rosario
Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of Quickchanges to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P100 (TEV, QC with O36/O37: P111, P112, P113) and P102 (EreB, QC with O16/O17: P106, P107, P108, P109, P110) were successful
Procedure:
Mastermix for analytical digestion of P106, P107, P108, P109, P110, P111, P112, P113 with AgeI-HF and XbaI:
volume
reagent
20 µl
NEBuffer 4 (10x)
2 µl
BSA (100x)
2.5 µl
AgeI-HF (20 U/µl)
2.5 µl
XbaI
148 µl
ddH2O
= 175 µl
TOTAL
17,5 µl Mastermix with 2,5 µl Plasmid
Mastermix for analytical digestion of P111, P112, P113 with XbaI and SpeI and P106, P107, P108, P109, P110 with XbaI and PstI-HF:
volume
reagent
20 µl
NEBuffer 4 (10x)
2 µl
BSA (100x)
2.5 µl
XbaI
148 µl
ddH2O
= 172,5 µl
TOTAL
17,25 µl Mastermix with 2,5 µl Plasmid and 0,25 µl SpeI or PstI respectively
Mastermix for analytical digestion of with P106, P107, P108, P109, P110 EcoRI and SpeI:
volume
reagent
12 µl
NEBuffer 4 (10x)
1,2 µl
BSA (100x)
1,5 µl
EcoRI-HF
1,5 µl
SpeI-HF
88.8 µl
ddH2O
= 105 µl
TOTAL
17,5 µl Mastermix with 2,5 µl Plasmid
Mastermix for analytical digestion of with P106, P107, P108, P109, P110 XbaI and SpeI:
volume
reagent
12 µl
NEBuffer 4 (10x)
1,2 µl
BSA (100x)
1,5 µl
XbaI
1,5 µl
SpeI-HF
88.8 µl
ddH2O
= 105 µl
TOTAL
17,5 µl Mastermix with 2,5 µl Plasmid
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
P111 XbaI + AgeI
P112 XbaI + AgeI
P113 XbaI + AgeI
P111 XbaI + SpeI
P112 XbaI + SpeI
P113 XbaI + SpeI
Ctrl
Ctrl
Ctrl
QC succesful
QC failed
QC succesful
1 kbp ladder DNA ladder
P106 XbaI + AgeI
P107 XbaI + AgeI
P108 XbaI + AgeI
P109 XbaI + AgeI
P110 XbaI + AgeI
P106 XbaI + PstI
P107 XbaI + PstI
P108 XbaI + PstI
P109 XbaI + PstI
P110 XbaI + PstI
Ctrl
Ctrl
Ctrl
Ctrl
Ctrl
PstI inside as should be; useless experiment
PstI inside as should be; useless experiment
PstI inside as should be; useless experiment
PstI inside as should be; useless experiment
PstI inside as should be; useless experiment
1 kbp ladder DNA ladder
P106 XbaI + SpeI
P107 XbaI + SpeI
P108 XbaI + SpeI
P109 XbaI + SpeI
P110 XbaI + SpeI
P106 EcoRI + SpeI
P107 EcoRI + SpeI
P108 EcoRI + SpeI
P109 EcoRI + SpeI
P110 EcoRI + SpeI
Ctrl
Ctrl
Ctrl
Ctrl
Ctrl
QC succesful
QC succesful
QC succesful
QC succesful
QC failed
Thursday, May 23rd
[Expand] Sequencing of P111, P113 and P109 (?)
Investigator: Jeff
Aim of the experiment: Sequencing of P111, P113 and P109 to check whether the QuickChange brought further unwanted mutations.
Procedure:
[Expand] PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV
Investigator: Jeff
Aim of the experiment: PCR of P111 (TEV S219V, SpeI-less) to generate Split-TEV. To generate N-TEV, the primer O38 and O39 were used. To generate C-TEV, the primer O40 and O41 were used.
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (O38 (N_Split_TEV_fw) for generating N-TEV; O40 (C_Split_TEV_fw) for generating C-TEV)
1 µl
10 µM Reverse Primer (O39 (N_Split_TEV_rv) for generating N-TEV; O41 (C_Split_TEV_rv) for generating C-TEV)
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P111)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme with following conditions:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
51 °C
60 s
68 °C
30 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Resulting product was labeled as F24 (N-TEV) and F25 (C-TEV).
[Expand] Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV)
Investigator: Jeff, Rosario
Aim of the experiment: Analytical gelelectrophoresis of F24 and F25 (N-TEV and C-TEV) to check whether PCR has worked.
Procedure:
4 µl of F24 and F25 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.
PCR products has expected length, but there are unspecific products. So we have to be cautious and have to check if we ligate the right products.
[Expand] Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI
Investigator: Jeff, Rosario
Aim of the experiment: Preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI to clone it into pSB1C3.
Procedure:
Batch for preparative digestion of PCR products (N-TEV and C-TEV) of P111 (TEV S219V, SpeI-less) with XbaI & AgeI.
volume
reagent
25 µl
PCR product F24/F25
5 µl
NEBuffer 4
0.5 µl
BSA (100x)
1 µl
AgeI-HF
1 µl
XbaI
17.5 µl
ddH2O
=50 µl
TOTAL
Incubation for 150 min at 37 °C.
Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
The digested fragments were labelled F26 (digestion of F24 with XbaI&AgeI) and F27 (digestion of F25 with XbaI&AgeI)
[Expand] Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Ligation of F17+F26 (N-TEV in pSB1C3) and F17+F27 (C-TEV in pSB1C3).
Procedure:
Ligation batch for F17+F26:
volume
reagent
1.63 µl
F17 (61.4 ng/µl, 2086 bp)
3.20 µl
F26 (15.9 ng/µl, 354 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
12.17 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F17+F27 (positive control)
volume
reagent
1.63 µl
F17 (61.4 ng/µl, 2086 bp)
2.41 µl
F27 (21.1 ng/µl, 354 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
12.96 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
[Expand] Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange (SDMI) Product of P28
Investigator: Jeff, Andi, Rosario, Johanna
Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange Product of P28. 8 clones were picked the day before.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and analytical gelelectrophoresis of P28, P117, P118, P119, P120, P121, P122, P123
Investigator: Rosario, Andi, Johanna, Jeff
Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P28 (promoter SV40) and quickchange products P117, P118, P119, P120, P121, P122, P123 (all QC - SDMI - of Bpul Laccase to remove forbidden AgeI).
Procedure:
Mastermix for analytical digestion of P28, P117, P118, P119, P120, P121, P122, P123 AgeI-HF:
volume
reagent
20 µl
NEBuffer 4 (10x)
2 µl
BSA (100x)
2.5 µl
AgeI-HF (20 U/µl)
150.5 µl
ddH2O
=175 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.
Results:
1 kbp ladder DNA ladder
Digestion of P28
Digestion of P116
Digestion of P117
Digestion of P118
Digestion of P119
Digestion of P120
Digestion of P121
Digestion of P122
Digestion of P123
as expected
QC successful
QC successful
QC successful
QC successful
QC successful
QC successful
QC successful
QC successful
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P28 (Laccase), P106 (EreB)
Investigator: Jeff, Andi, Johanna, Rosario
Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P106 (EreB).
Procedure: Quickchange - PCR
Reaction batch - P106
volume
reagent
Additional information
5 µl
10x Pfu Ultra II buffer
2 µl
Plasmid template (P106 - 1:10 dilution)
need to get to 50 ng/µl
1.25 µl
1:10 dilution of O18 (for P106, 10 µM)
1.25 µl
1:10 dilution of O19 (for P106, 10 µM)
38.5 µl
ddH2O
1 µl
dNTP mix
1 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P106
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
18
95 °C
30 s
52 °C
1 min
68 °C
4 min
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
Reaction batch - P116 (Laccase)
volume
reagent
Additional information
5 µl
10x Pfu Ultra II buffer
2 µl
Plasmid template - 1:5 dilution
need to get to 50 ng/µl
1.25 µl
1:10 dilution of O28 for P116
1.25 µl
1:10 dilution of O29 for P116
38.5 µl
ddH2O
1 µl
dNTP mix
1 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P116
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
18
95 °C
30 s
52 °C
1 min
68 °C
4 min
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue
Investigator: Jeff, Andi, Johanna, Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P106 and P116.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Friday, May 24th
[Expand] Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)
Investigator: Jeff, Florian
Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions was plated again on a new chlorampenicol plates.
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P116 (Laccase), P109 (EreB)
Investigator: Volker, Louise, Katrin, Flo
Aim of the experiment: Removal of forbidden restiction sites from P116 (Laccase), P109 (EreB).
Procedure: Quickchange - PCR
Reaction batch - P109
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P109 - 1:10 dilution)
need to get to 50 ng/µl
1 µl
1:10 dilution of O18 (for P109, 10 µM)
1 µl
1:10 dilution of O19 (for P109, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P109
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
21
95 °C
30 s
52 °C
1 min
68 °C
4 min 50 s
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
Reaction batch - P116 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template - 1:5 dilution
need to get to 50 ng/µl
1 µl
1:10 dilution of O28 for P116
1 µl
1:10 dilution of O29 for P116
18.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P116
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
21
95 °C
30 s
52 °C
1 min
68 °C
4 min 50 s
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Transformation of the Quickchange products into E. coli XL1 blue
Investigator: Volker
Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P109 and P116.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Saturday, May 25th
[Expand] Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful
Investigator: Volker, Louise, Katrin, Flo
Aim of the experiment: Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful
Procedure:
5 µl of the PCR Quickchange products were either mixed with with 0.5 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.
[Expand] Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)
Investigator: Volker
Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).
Procedure:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
5 colonies for P116QC (Laccase, QC with O28/O29) and 6 colonies for P109QC (EreB, QC with O18/O19) were picked from the plates.
[Expand] Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3)
Investigator: Volker
Aim of the study: Picking of E. coli XL1 blue with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).
Procedure:
Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)
5 colonies were picked from plates for E. colis XL1 blue transformed with ligation products F17+F26 (N-TEV S219V in pSB1C3) and F17+F27 (C-TEV S219V in pSB1C3).
Sunday, May 26th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)
Investigator: Katrin, Louise
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19) were successful
Investigator: Katrin, Louise
Aim of the experiment:Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)
Procedure:
analytical digestion of Quickchange products of P109 (EreB, QC with O18/O19)
volume
reagent
2 µl
NEB CutSmart Buffer (10x)
0,25 µl
PstI-HF (20 U/µl)
2,5 µl
DNA from Miniprep
15,25 µl
ddH2O
=20 µl
TOTAL
in order to check if the QC was successful, P109 (before QC) was also digested
analytical digestion of Quickchange products of P116 (Laccase, QC with O28/O29)
volume
reagent
2 µl
NEB CutSmart Buffer (10x)
0,25 µl
EcoRI-HF
0,25 µl
NgoMIV
2,5 µl
DNA from Miniprep
15 µl
ddH2O
=20 µl
TOTAL
in order to check if the QC was successful, P116 (before QC) was also digested
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder#
P116 (Laccase before QCII) digested with EcoRI-HF, NgoMIV
Laccase after QCII (P128) digested with EcoRI-HF, NgoMIV
Laccase after QCII (P129) digested with EcoRI-HF, NgoMIV
Laccase after QCII (P130) digested with EcoRI-HF, NgoMIV
Laccase after QCII (P131) digested with EcoRI-HF, NgoMIV
Laccase after QCII (P124) digested with EcoRI-HF, NgoMIV
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
as expected
as expected
as expected
as expected
as expected
as expected
Laccase P124 was chosen for sequencing
1 kbp ladder DNA ladder
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
EreB after QCII (P125) digested with PstI
EreB after QCII (P132) digested with PstI
EreB after QCII (P133) digested with PstI
EreB after QCII (P134) digested with PstI
EreB after QCII (P135) digested with PstI
EreB after QCII (P136) digested with PstI
EreB before QCII (P109) digested with PstI
as expected
as expected
as expected
as expected
as expected
as expected
as expected
EreB P125 was chosen for sequencing
[Expand] Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3
Investigator: Katrin, Louise
Aim of the experiment: Miniprep of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25) with pSB1C3
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of ligation products F17 + F26, F17 + F27
Investigator: Katrin, Louise
Aim of the experiment: Analytical digestion and gelelectrophoresis to check whether the ligation of N-TEV (F17+F26) and C-TEV (F17+F25) in pSB1C3 were successful
Procedure:
analytical digestion of ligation products of N-TEV (F17+F26) and C-TEV (F17+F25)in pSB1C3
Mastermix:
volume
reagent
22 µl
NEB Buffer 4 (10x)
2,2 µl
BSA (100X)
2,75 µl
XbaI
2,75 µl
AgeI-HF
162,8 µl
ddH2O
=192,5 µl
TOTAL
2,5 µl of miniprep products were added to 17,5 µl Mastermix
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder#
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
N-TEV ligated into pSB1C3 (F17 + F26) (P126) digested with XbaI, AgeI-HF
N-TEV ligated into pSB1C3 (F17 + F26) (P137) digested with XbaI, AgeI-HF
N-TEV ligated into pSB1C3 (F17 + F26) (P138) digested with XbaI, AgeI-HF
N-TEV ligated into pSB1C3 (F17 + F26) (P139) digested with XbaI, AgeI-HF
as expected
as expected
as expected
as expected
N-TEV ligated into pSB1C3 (F17 + F26) (P126) was chosen for sequencing
1 kbp ladder DNA ladder
C-TEV ligated into pSB1C3 (F17 + F27) (P127) digested with XbaI, AgeI-HF
C-TEV ligated into pSB1C3 (F17 + F27) (P140) digested with XbaI, AgeI-HF
C-TEV ligated into pSB1C3 (F17 + F27) (P141) digested with XbaI, AgeI-HF
C-TEV ligated into pSB1C3 (F17 + F27) (P142) digested with XbaI, AgeI-HF
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
belongs to other experiment
as expected
as expected
as expected
as expected
C-TEV ligated into pSB1C3 (F17 + F27) (P127) was chosen for sequencing
[Expand] Sequencing of P124-P127
Investigators: Louise, Katrin
Aim of the experiment: Sequencing of QCII products (EreB, Laccase) and ligation product of split-TEV
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
p124
Miniprep of transformation of QC product of P116 (Laccase, QC with O28/O29)(QC - SDMII - of Bpul Laccase to remove forbidden NgoMIV)
FR01002337 (VR)
FR01002338 (VF2)
p125
Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)
FR01002339 (VR)
FR01002340 (VF2)
p126
Miniprep of ligation product F17 + F26 (N-TEV)
FR01002341 (VR)
FR01002342 (VF2)
p127
Miniprep of ligation product F17 + F27 (C-TEV)
FR01002343 (VR)
FR01002344 (VF2)
Week 6
Monday, May 27th
[Expand] Oligohybridization of MiniMCS (O56,O57) and Spytag (O54,O55)
Investigator: Johanna
Aim of the experiment: Oligohybridization of MiniMCS (MiniMCSII_fw (O56) and MiniMCSII_rv (O57) and of Spytag (SpyTag_fw (O54) and SpyTag_rv (O55))
Operational sequence
25 µL of 100 pmol/µl of MiniMCS fw and 25 µL of 100 pmol/µl MiniMCS rev in one Eppi. 25 µL of 100 pmol/µl of Spytag fw and 25 µL of 100 pmol/µl Spytag rev in a second Eppi
Heating up to 95 °C for 5 min
Cooling at RT in a styropor box overnight
[Expand] PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114)
Investigator: Johanna
Aim of the experiment: PCR of npt-Kasette (P115), PIF3 (P51), pActin (P114) and t35S (P114).
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P114; P115; P51)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
49 °C
60 s
68 °C
100 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Results:
PCR did not work properly because primer concentration was wrong.
[Expand] Preparative digestion and gelelectrophoresis of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI
Investigator: Andi
Aim of the experiment:Preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI to clone fragment of P45 into F17 (pSB1C3) and to fusion Middle linker of P39 with GFP of P20.
Procedure:
Batch for preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeIwith XbaI & AgeI.
volume
reagent
20 µl
P45
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
2 µl
AgeI-HF
2 µl
XbaI
11.6 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.
volume
reagent
20 µl
P39
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
2 µl
AgeI-HF
2 µl
PstI
11.6 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.
volume
reagent
20 µl
P45
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
2 µl
NgoMIV
2 µl
PstI
11.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 120 min at 37 °C.
Preparative gelelectrophoresis was performed at 90 V for 1.5 h.
1 kbp ladder
P20 digested with NgoMIV&PstI (=F30)
P20 digested with NgoMIV&PstI (=F30)
P39 digested with AgeI&PstI (=F29)
P39 digested with AgeI&PstI (=F29)
P45 digested with XbaI&AgeI (=F28)
P45 digested with XbaI&AgeI (=F28)
lower band was cut out
lower band was cut out
band was cut out
band was cut out
lower band was cut out
lower band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124(Laccase), P125 (EreB)
Investigator: Andi
Aim of the experiment: Removal of forbidden restiction sites from P124 (Laccase), P125 (EreB).
Procedure: Quickchange - PCR
Reaction batch - P124
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P124 - 1:6 dilution)
need to get to 50 ng/µl
1 µl
1:10 dilution of O30 (for P125, 10 µM)
1 µl
1:10 dilution of O31 (for P125, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch - P125
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P125 - 1:6 dilution)
need to get to 50 ng/µl
1 µl
1:10 dilution of O20 (for P125, 10 µM)
1 µl
1:10 dilution of O21 (for P125, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters for both batches
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
21
95 °C
30 s
52 °C
1 min
68 °C
4 min 50 s
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Preparation of ordered primers
Investigators: Andi
Aim of the experiment: Dissolving of primer pallets to provide a concentration of 100 pmol/µl
Procedure: We added an amount of water which gave us a final primer concentration of 100 pmol/µl.
Primers were labeled as follows:
Label
Oligonr.
O54
Spytag_fw
O55
Spytag_rev
O56
MiniMCSII_fw
O57
MiniMCSII_rev
O58
Npt_for
O59
Npt_rev
O60
t35S_for
O61
t35S_rev
O62
pACT_for
O63
pACT_rev
O64
NucA_for
O65
NucA_rev
O66
Pif3_for
O67
Pif3_rev
[Expand] Transformation of P45, P51, P100 and P102 into E. coli XL1 blue
Investigator: Andi, Johanna
Aim of the experiment: Transformation of E. coli XL1 blue with P45, P51, P102 and P100.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Picking of transformed Plasmid E. coli XL1 blue with P114, P115
Investigator: Andi
Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P114, P115.
Procedure:
Picking and overnight culture after standard laboratory's protocol. (AmpR LB-medium)
5 colonies of P114 and P115 were picked from the plates.
[Expand] Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3)
Investigator: Jeff
Aim of the experiment: Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3).
Procedure:
Ligation batch for F29+F30:
volume
reagent
4.22 µl
F29 (23.7 ng/µl, 2110 bp)
10.48 µl
F30 (9.5 ng/µl, ~700 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
2.3 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F17+F28:
volume
reagent
1.63 µl
F17 (61.4 ng/µl, 2086 bp)
7.8 µl
F28 (9.6 ng/µl, 522 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
7.57 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
Tuesday, May 28th
[Expand] Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67)
Investigator: Jeff, Rosario
Aim of the experiment: Analytical gelelectrophoresis of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67).
Procedure:
4 µl of PCR products of P114 (O62&O63), P114 (O60&O61), P115 (O58&O59), P51 (O66&O67) were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.
Wrong buffer (GC buffer, instead of standard reaction buffer, was used and 10x too much primers were also used)
[Expand] Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 into E. coli XL1 blue
Investigator: Andi, Johanna
Aim of the experiment: Transformation of Quickchangeproduct SDMIII of P124 (Laccase) and P125 (EreB) + ligation product of F29+F30 + ligation product of F17+F19 + ligation product of F17+NK + ligation product of F29+NK into E. coli XL1 blue.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Sequencing of P133 and P134
Investigators: Louise, Johanna
Aim of the experiment: Sequencing of two more QCII products (EreB)
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
p133
Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)
FR01002287 (fw)
FR01002296 (rev)
p134
Miniprep of transformation of QC product of P109 (EreB, QC with O18/O19) (QC - SDMII - of EreB to remove forbidden PstI)
FR01002295 (fw)
FR01002294 (rev)
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115
Investigator: Andi
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P114 and P115.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102
Investigator: Jeff, Rosario
Aim of the experiment: Picking of of E. coli XL1 blue transformed with P45, P51, P100, P102.
Procedure:
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (P51, P100, P102)/4 µL Ampicillin (1000x) (P45).
1 colony for each plasmid were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
[Expand] PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143)
Investigator: Rosario, Jeff
Aim of the experiment: PCR of npt-Kasette (P144), PIF3 (P51), pActin (P143) and t35S (P143).
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P114; P115; P51)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
47 °C
60 s
68 °C
120 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
[Expand] Analytical gelelectrophoresis of F33, F34, F35, F36
Investigator: Jeff, Rosario
Aim of the experiment: Analytical gelelectrophoresis of F33, F34, F35, F36.
Procedure:
4 µl of F33, F34, F35 and F36 were mixed with seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 40 min at 90 V.
1 kbp ladder
F33
F33
F33
F33
PCR did not work, maybe high GC content?
PCR products has expected length, some byproducts
PCR products has expected length, some byproducts
PCR products has expected length
Wednesday, May 29th
[Expand] Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18
Investigator: Andi
Aim of the experiment: Transformation of E. coli XL1 blue with SDMIII product of P124, ligation product of F29+F30 and ligation product of F17+F18.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45
Investigator: Rosario
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of P100, P102, P51, P45 .
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Picking of of E. coli XL1 blue transformed with ligation product of F17+F28
Investigator: Andi
Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product of F17+F28.
Procedure:
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) (
3 colonies were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P133 (EreB)
Investigator: Louise
Aim of the experiment: Removal of forbidden restiction site from P133 (EreB).
Procedure: Quickchange - PCR
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P133 - 1:10 dilution)
need to get to 50 ng/µl
1 µl
1:10 dilution of O20 (for P133, 10 µM)
1 µl
1:10 dilution of O21 (for P133, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P133
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
3 min 30 s
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.
[Expand] Transformation of the Quickchange product into E. coli XL1 blue
Investigator: Louise
Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM III of P133.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI
Investigator: Louise
Aim of the experiment: Preparative digestion of t35s(F34) with XbaI & PstI, npt(F25) with EcoRI & SpeI, PIF3(F36) with XbaI & AgeI-HF and P10 with XbaI & PstI as well as with EcoRI & SpeI.
Procedure:
Batch for preparative digestion of t35s(F34) with XbaI and PstI
volume
reagent
25 µl
Plasmid DNA P61
5 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of npt(F35) with EcoRI and SpeI
volume
reagent
25 µl
Plasmid DNA P61
5 µl
CutSmart Buffer
1 µl
EcoRI (20 U/µl)
1 µl
SpeI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of PIF3(F36) with XbaI and AgeI-HF
volume
reagent
25 µl
Plasmid DNA P61
5 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
1 µl
AgeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P10 with XbaI & PstI for backbone isolation.
volume
reagent
20 µl
Plasmid DNA P96
4 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P10 with EcoRI and SpeI for backbone isolation.
volume
reagent
20 µl
Plasmid DNA P96
4 µl
CutSmart Buffer
1 µl
EcoRI (20 U/µl)
1 µl
SpeI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
The resulting fragments were named: ?????
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 90 min.
1 kbp ladder DNA ladder
Digestion of PCR product t35s (F34) with XbaI & PstI = F38
Digestion of PCR product npt casette (F25) with EcoRI & SpeI = F39
Digestion of PCR product PIF3 (F36) with XbaI & AgeI-HF = F40
as expected, lower band was cut out
as expected, bright band was cut out
as expected, bright band was cut out
1 kbp ladder DNA ladder
Digestion of P10 with EcoRI & SpeI = F41
Digestion of P10 with XbaI & PstI = F42
as expected, lower band was cut out
as expected, lower band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030)
Investigator: Florian, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3 containing a NLS from SV40 (BBa_K801030).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] PCR of pActin (P143)
Investigator: Florian, Jeff
Aim of the experiment: PCR of pActin (P143).
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P143:O62 (pActin_for))
1 µl
10 µM Reverse Primer (P143:O63 (pActin_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P143)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
51 °C
60 s
68 °C
80 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Results:
After analytical gelelectrophoresis there was no product visible. PCR did't work!
[Expand] Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))
Investigator: Jeff
Aim of the experiment: Ligation of F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).
Procedure:
Ligation batch for F17+F40:
volume
reagent
1.63 µl
F17 (61.4 ng/µl, 2086 bp)
1.88 µl
F40 (22.8 ng/µl, ~297 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
13.49 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F42+F38:
volume
reagent
1.36 µl
F42 (73.5 ng/µl, 2070 bp)
3.42 µl
F38 (9.2 ng/µl, 217 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
12.22 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F41+F39:
volume
reagent
1.11 µl
F41 (89.9 ng/µl, 2070 bp)
13.74 µl
F39 (16.0 ng/µl, 1517 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
2.15 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
Thursday, May 30th
[Expand] Gradient PCR of pActin (P143)
Investigator: Ingmar
Aim of the experiment: PCR of pActin
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Reaction Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer O62 (pActin_for)
1 µl
10 µM Reverse Primer O63 (pActin_rev)
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA P143
35.75 µL
ddH2O Water
=50 µL
TOTAL
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq GC Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer O62 (pActin_for)
1 µl
10 µM Reverse Primer O63 (pActin_rev)
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA P143
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
47 °C
60 s
68 °C
120 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
A temperature gradient was applied beginning at 47°C and ranging to 51°C. For each integer (47, 48, 49, 50 and 51°C) a single PCR batch was used.
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Analytical gelelectrophoresisProcedure:
4 µl of each PCR product were mixed seperately with 0.44 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.
1 kbp ladder
F45 GC buffer
F46 GC buffer
F47 GC buffer
F47 GC buffer
F49 GC buffer
normal buffer
normal buffer
normal buffer
normal buffer
normal buffer
1 kbp ladder
PCR products has expected length
PCR products has expected length
PCR products has expected length
PCR products has expected length
PCR products has expected length
PCR products has expected length, some byproducts
PCR products has expected length, some byproducts
PCR products has expected length, some byproducts
PCR products has expected length, some byproducts
PCR products has expected length, some byproducts
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25)
Investigator: Jeff, Flo
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue of F17+F28 (FluA in pSB1C3 RFC25).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25)
Investigator: Flo, Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of ligation products F17+F28 (FluA in pSB1C3 RFC25).
Procedure:
Analytical digestion of ligation products of F17+F28 (FluA in pSB1C3 RFC25) with XbaI & AgeI.
volume
reagent
2.5 µl P150/P151/P152/P153
2 µl
CutSmart Buffer (10x)
0.25 µl
XbaI
0.25 µl
AgeI-HF
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder#
Digestion of P150 with XbaI&AgeI-HF
Digestion of P151 with XbaI&AgeI-HF
Digestion of P152 with XbaI&AgeI-HF
Digestion of P153 with XbaI&AgeI-HF
as expected
as expected
as expected
as expected
[Expand] Preparative gelelectrophoresis of oligohybridization products F31 & F32
Investigator: Flo, Jeff
Aim of the experiment: Preparative gelelectrophoresis of oligohybridization products F31 & F32.
Procedure:
50 µl of the 1:10 dilution of F31 and F32 were mixed with 5 µl of DNA loading buffer (10x).
Preparative gelelectrophoresis was performed 1.5 h on a 2% agarose gel at 90 V.
1 kbp ladder
F31
F32
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Gelpurified F31 and F32 were labelled as F43 and F44.
[Expand] Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10))
Investigator: Florian, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with F17+F40 (PIF3 in pSB1C3(RFC25)), F42+F38 (t35S in pSB1C3(RFC10)) and F41+F39(npt-casette in pSB1C3(RFC10)).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.
[Expand] Ligation of F42+F43 and F17+F32
Investigator: Rosario
Aim of the experiment: Ligation of F42+F43 and F17+F32.
Procedure:
Ligation batch for F42+F43:
volume
reagent
1.36 µl
F42 (73.5 ng/µl, 2070 bp)
1.51 µl
F43 (5.4 ng/µl, 34 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
14.13 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F17+F32:
volume
reagent
1.63 µl
F17 (61.4 ng/µl, 2086 bp)
0.25 µl
F32 (2510.8 ng/µl, 49 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
15.12 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature&
[Expand] Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS)
Investigator: Louise
Aim of the experiment: Picking of of E. coli XL1 blue transformed with EreB SDMIII (P149) and BBa_K801030 (SV40 NLS).
Procedure:
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
5 colonies of EreB SDMIII (P149) and 2 colonies of BBa_K801030 (SV40 NLS) were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
Friday, May 31st
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)
Investigator: Louise, Jeff, Katrin
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P149 (QCIII of EreB) and BBa_K801030 (SV40 NLS)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of P149 (QCIII of EreB) with EcoRI-HF and PstI-HF
Investigator: Louise, Jeff, Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF to check if QCII was successful
Procedure:
Analytical digestion of P154-P158 (QCIII of EreB) with EcoRI-HF and PstI-HF
volume
reagent
2.5 µl
P149-P158/P133 (control before QCIII)
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder#
Digestion of P133 with EcoRI-HF and PstI-HF
Digestion of P154 with EcoRI-HF and PstI-HF
Digestion of P155 with EcoRI-HF and PstI-HF
Digestion of P156 with EcoRI-HF and PstI-HF
Digestion of P157 with EcoRI-HF and PstI-HF
Digestion of P158 with EcoRI-HF and PstI-HF
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Sequencing of P157 and P160
Investigators: Louise, Jeff, Katrin
Aim of the experiment: Sequencing of QCIII product (EreB) and BBa_K801030 (SV40 NLS)
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
P157
Miniprep of transformation of QCIII product of P149 (QC - SDMIII - of EreB to remove forbidden PstI)
FR01002289 (VR)
FR01002290 (VF2)
P160
Miniprep of transformation of BBa_K801030 (SV40 NLS)
FR01002291 (VR)
FR01002292 (VF2)
[Expand] Ligation of F29+F30
Investigator: Katrin
Aim of the experiment: Ligation of F29+F30
Procedure:
Ligation batch
volume
reagent
4.22 µl
F29 (23.7 ng/µl, 2110 bp))
10.48 µl
F30 (9,5 ng/µl, about 700 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
2.3 µl
ddH2O
=20 µl
TOTAL
No negative control was prepared because there was not enough vector backbone left
Ligation was performed at 37 °C overnight
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124 (Laccase)
Investigator: Louise, Katrin
Aim of the experiment: Removal of forbidden restiction site from P124 (Laccase).
Procedure: Quickchange - PCR
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P124 - 1:10 dilution)
need to get between 5 and 50 ng/µl
1 µl
1:10 dilution of O30 (for P124, 10 µM)
1 µl
1:10 dilution of O31 (for P124, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P124
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
4 min
4 °C
hold
After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour
[Expand] Preparative digestion of pActin(F46) with XbaI & PstI
Investigator: Ingmar
Aim of the experiment: Preparative digestion of pActin(F46) in order to ligat this fragment in pSB1C3 later on.
Procedure:
Batch for preparative digestion of pActint(F46) with XbaI and PstI
volume
reagent
25 µl
F46 PCR product of pActin
5 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batchesand loaded on a 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 90 min.
The band was extracted by QIAquick Gel Extraction Kit, QIAGEN. The resulting fragment was named: F50
[Expand] Ligation of F42+F50 (pActin in pSB1C3)
Investigator: Ingmar
Aim of the experiment: Ligation of F42+F50 (pActin in pSB1C3(RFC25).
Procedure:
Ligation batch
volume
reagent
1.37 µl
F42
13.13 µl
F50
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
2.5 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
[Expand] Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt)
Investigator: Ingmar
Aim of the experiment: Picking of of E. coli XL1 blue transformed with F42+F38 (t35S), F17+F40 (Pif 3) and F41+F39 (npt).
Procedure:
The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 5 mL of LB-medium + 5 µL chloramphenicol(1000x).
3 colonies of each agar plate were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
[Expand] Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32
Investigator: Louise, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F42+F43 and F17+F32.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
Saturday, June 1st
[Expand] Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F29+F30 (GFP in pSB1C3), F42+F50 (actin promoter in pSB1C3), F42+F50 (negative control)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.
[Expand] Transformation of E. coli XL1 blue with QCIII product of Laccase (SDMIII of P124)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1 blue with QCIII product of Laccase
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on a new chlorampenicol plates.
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)
Investigator: Katrin
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F39 + F41 (npt-casette in pSC1C3),F42+F38 (t35s in pSB1C3), F17+F40 (PIF3 in pSB1C3)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSB1C3), P164-166 (F42+F38, t35s in pSB1C3), P167-169 (F17+F40, PIF3 in pSB1C3)
Investigator: Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P161-163 (F39 + F41, npt-casette in pSC1C3) with EcoRI-HF and SpeI-HF, P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF and P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI
Procedure:
Analytical digestion of P161-163 (F39 + F41, npt-casette in pSC1C3), P164-166 (F42+F38, t35s in pSB1C3) with EcoRI-HF and SpeI-HF
volume
reagent
2.5 µl
P161-P166
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI-HF
0.25 µl
SpeI-HF
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Analytical digestion of P167-169 (F17+F40, PIF3 in pSB1C3) with AgeI-HF and XbaI
volume
reagent
2.5 µl
P167-P169
2 µl
CutSmart Buffer (10x)
0.25 µl
AgeI-HF
0.25 µl
XbaI
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder#
Digestion of P161 with EcoRI-HF and SpeI-HF
Digestion of P162 with EcoRI-HF and SpeI-HF
Digestion of P163 with EcoRI-HF and SpeI-HF
Digestion of P164 with EcoRI-HF and SpeI-HF
Digestion of P165 with EcoRI-HF and SpeI-HF
Digestion of P166 with EcoRI-HF and SpeI-HF
Digestion of P167 with AgeI-HF and XbaI
Digestion of P168 with AgeI-HF and XbaI
Digestion of P169 with AgeI-HF and XbaI
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
Sunday, June 2nd
[Expand] Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F44 and F42+F32
Investigator: Andi
Aim of the experiment: Picking of of E. coli XL1 blue transformed with QCIII product of P124 and the ligation products of F17+F32 and F42+F32.
Procedure:
The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
4 colonies of each agar plate were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
Week 7
Monday, June 3rd
[Expand] Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10)
Investigator: Andi, Johanna, Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10), P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10).
Procedure:
Mastermix for analytical digestion of P170-P173 (F17+F32, SpyTag in pSB1C3 RFC25), P174-P177 (F42+F43, miniMCSII in pSB1C3 RFC10) with XbaI+PstI.
volume
reagent
18 µl
CutSmart Buffer (10x)
2.25 µl
EcoRI-HF
2.25 µl
SpeI-HF
135 µl
ddH2O
=157.5 µl
TOTAL
17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P170-P177)
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Analytical digestion of P178-181 (QCIII von P142, Laccase in pSB1C3 RFC10):
volume
reagent
2.5 µl
P178-P181
2 µl
CutSmart Buffer (10x)
0.25 µl
NgoMIV
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder
Digestion of P170 with EcoRI-HF and PstI-HF
Digestion of P162 with EcoRI-HF and PstI-HF
Digestion of P163 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
Digestion of P164 with EcoRI-HF and PstI-HF
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
as expected, insert too small to be visible
1 kbp ladder DNA ladder
Digestion of P142 with NgoMIV and PstI-HF
Digestion of P178 with NgoMIV and PstI-HF
Digestion of P179 with NgoMIV and PstI-HF
Digestion of P180 with NgoMIV and PstI-HF
Digestion of P181 with NgoMIV and PstI-HF
Ctrl (before QuickChange III)
as expected, plasmid is only linearized, all NgoMIV sites are removed
as expected, plasmid is only linearized, all NgoMIV sites are removed
as expected, plasmid is only linearized, all NgoMIV sites are removed
as expected, plasmid is only linearized, all NgoMIV sites are removed
Tuesday, June 4th
[Expand] Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag)
Investigators: Louise, Johanna
Aim of the experiment: Sequencing of P162(npt-cassete), P165(t35s), P168(PIF3), P179(LaccQCIII), P177(MiniMCS), P170(Spytag).
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode1
Barcode2
P162
Miniprep of ligation product F39 + F41 (npt-casette in pSB1C3)
FR01002331 (VR)
FR01002330 (VF2)
P165
Miniprep of ligation product F42+F38 (t35s in pSB1C3)
FR01002332 (VF2)
P168
Miniprep of ligation product F17+F40 (PIF3 in pSB1C3)
FR01002333 (VF2)
P179
Miniprep of ligation product Laccase QCIII (P142)
FR01002335 (VR)
FR01002334 (VF2)
P177
Miniprep of ligation product MiniMCSII F42+F43
FR01002336 (VF2)
P170
Miniprep of ligation product F17+F32 (SpyTag)
FR01002328 (VF2)
[Expand] Preparative digestion and gelelectrophoresis of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI
Investigator: Louise, Johanna
Aim of the experiment: Preparative digestion of P39(middle-linker) with PstI and AgeI-HF, P20(GFP-Psb1C3) with NgoMIV and PstI, F45(pActin) with XbaI and PstI.
Procedure:
Batch for preparative digestion of P39(middle-linker) with PstI and AgeI-HF
volume
reagent
15 µl
Plasmid DNA P39
4 µl
CutSmart Buffer
1 µl
AgeI-HF(20 U/µl)
1 µl
PstI (20 U/µl)
19 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P20(GFP-Psb1C3) with NgoMIV and PstI
volume
reagent
20 µl
Plasmid DNA P20
4 µl
CutSmart Buffer
1 µl
NgoMIV (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of F45(pActin) with XbaI and PstI
volume
reagent
25 µl
Fragment DNA F45
5 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 90 min.
1 kbp DNA ladder
Digestion of P20 with NgoMIV & PstI = F53
Digestion of P39 with AgeI & PstI = F52
Digestion of F45 with XbaI & PstI = F51
as expected, lower band was cut out
as expected, band was cut out
as expected, lower band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Quick Change mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)
Investigator: Andi, Jeff
Aim of the experiment: Removal of forbidden restiction site SDM IV from P179 (Laccase).
Procedure: Quickchange - PCR
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P179 - 1:6 dilution)
need to get between 5 and 50 ng/µl
1 µl
1:10 dilution of O32 (for P179, 10 µM)
1 µl
1:10 dilution of O33 (for P179, 10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters - P179
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
4 min
4 °C
hold
After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour
[Expand] Preparative digestion and gelelectrophoresis of P182 (Gensynthesis 1) with XbaI and AgeI-HF, P183 (Gensynthesis 2) with XbaI and AgeI-HF
Investigator: Andi, Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P182 (Gensynthese 1) with XbaI and AgeI-HF, P183 (Gensynthese 2) with XbaI and AgeI-HF
Procedure:
Batch for preparative digestion of P182 with XbaI and AgeI-HF
volume
reagent
15 µl
Plasmid DNA P182
4 µl
CutSmart Buffer
1 µl
AgeI-HF(20 U/µl)
1 µl
XbaI (20 U/µl)
19 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P183 with XbaI and AgeI- HF
volume
reagent
15 µl
Plasmid DNA P183
4 µl
CutSmart Buffer
1 µl
AgeI-HF (20 U/µl)
1 µl
XbaI (20 U/µl)
19 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1.5% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 70 V for 60 min.
1 kbp DNA ladder
Digestion of Gensynthesis 1 with XbaI & AgeI
100 bp DNA ladder
Digestion of Gensynthesis 2 with XbaI & AgeI
1 kbp DNA ladder
as expected, fragments (78 bp and 530 bp) were cut out
as expected, fragments (111 bp, 206 bp and 359 bp) were cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF
Investigator: Andi, Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P9 with XbaI and AgeI-HF
Procedure:
Batch for preparative digestion of P9 with XbaI and AgeI-HF
volume
reagent
15 µl
Plasmid DNA P9
4 µl
CutSmart Buffer
1 µl
AgeI-HF(20 U/µl)
1 µl
XbaI (20 U/µl)
19 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
The resulting fragments were named: ?????
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 70 V for 60 min.
Gelfragmentation was like expected, both bands were cut out (upper band = PhyB, lower band = pSB1C3 RFC25)
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase)
Investigator: Andi, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with Quickchange product SDM IV of P179 (Laccase).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2x 10 µl of DNA was added seperately into 2x 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
[Expand] Transformation of E. coli XL1 blue with P39
Investigator: Louise, Johanna
Aim of the experiment: Transformation of E. coli XL1 blue with P39
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added seperately into 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
[Expand] Preparative digestion and gelelectrophoresis of P39 with AgeI & PstI, P20 with NgoMIV & PstI
Where is the snapshot of the gel =DDDD
Investigator: Johanna, Louise
Aim of the experiment:Preparative digestion of P39 with AgeI & PstI, P20 with NgoMIV & PstI to fusion Middle linker of P39 with GFP of P20.
Procedure:
Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.
volume
reagent
20 µl
P39
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
2 µl
AgeI-HF
2 µl
PstI
11.6 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.
volume
reagent
20 µl
P45
4 µl
NEBuffer 4
0.4 µl
BSA (100x)
2 µl
NgoMIV
2 µl
PstI
11.6 µl
ddH2O
=40 µl
TOTAL
Incubation for 150 min at 37 °C.
Preparative gelelectrophoresis was performed at 90 V for 1 h.
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51
Investigator: Jeff, Andi, Johanna, Louise
Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.
Procedure:
Ligation batch for F54+F59 :
volume
reagent
2.2 µl
F54 (5.9 ng/µl, 90 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
13.3 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F55+F59:
volume
reagent
11.6 µl
F55 (6.4 ng/µl, 520 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
3.9 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F56+F59:
volume
reagent
3.3 µl
F56 (4.7 ng/µl, 110 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
12.2 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F57+F59:
volume
reagent
3.1 µl
F57 (9.1 ng/µl, 200 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
12.4 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F58+F59:
volume
reagent
4.4 µl
F58 (12.9 ng/µl, 400 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
11.1 µl
ddH2O
=20 µl
TOTAL
Negative Control Ligation batch for F59:
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
15.5 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F52+F53:
volume
reagent
10 µl
F52 (9.9 ng/µl, XXX bp)
7 µl
F53 (12.2 ng/µl, XXX bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F42+F51:
volume
reagent
1.36 µl
F42 (XXX ng/µl, XXX bp)
15.64 µl
F51 (8.2 ng/µl, 1237 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Cycled ligation has been performed after following protocol in a thermocycler:
99 cycles
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
12 °C
60 s
22 °C
60 s
Hold
16 °C
infinite
Lid temperature = 37 °C
[Expand] Plating of received E. coli containing biobricks
Investigator: Jeff, Andreas
Aim of the experiment: The received biobricks were already transformed in E. coli and were in an agar stabs. These E. coli cells were transferred with an inoculation loop on antibiotic selection plates and were incubated over night.
Operational sequence:
Bacterias containing plasmids with biobricks were transferred with a sterile inoculation loop on antibiotic plates and were incubated at 37 °C overnight.
The biobricks were:
Name
Function
BBa_J61032
Alkaline Phosphatase
BBa_K426020
Self lysis device
BBa_K729004
Nuclease from S. Aureus
BBa_K365001
PIF6
BBa_K648011
RBS B0034 with cI repressor C0051 under the control of constitutive promoter J23113
Wednesday, June 5th
[Expand] Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53
Investigator: Florian, Rosario, Louise, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue transformed with F59+F54, F59+F55, F59+F56, F59+F57, F59+F58, F42+F51 and F52+F53.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics.
[Expand] Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020)
Investigator: Rosario, Florian, Louise, Jeff
Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_K365001 (PIF6), BBa_K729004 (Nuclease from Staphylococcus aureus), BBa_K648011 (XylE RFC25), BBa_J61032 (Alkaline phosphotase), BBa_K426020 (BBa_K426020).
Procedure:
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampicillin(1000x).
2 colonies of each plate were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight.
Thursday, June 6th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011
Investigator: Ingmar
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P39, P20, genesynthesis 1&2 and BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
P39
P184
P39
P185
P20
P186
P20
P187
BBa_J61032
P188
BBa_J61032
P189
BBa_K426020
P190
BBa_K426020
P191
BBa_K729004
P192
BBa_K729004
P193
BBa_K365001
P194
BBa_K365001
P195
BBa_K648011
P196
BBa_K648011
P197
Genesynthesis 2
P198
Genesynthesis 2
P199
Genesynthesis 1
P200
Genesynthesis 1
P201
[Expand] Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011)
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P188-P197 (latest received biobricks: BBa_J61032, BBa_K426020, BBa_K729004, BBa_K365001, BBa_K648011).
Procedure:
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI-HF
0.25 µl
SpeI-HF
15 µl
ddH2O
2.5 µl
Plasmid
=20 µl
TOTAL
Mastermix (12x)
volume
reagent
24 µl
CutSmart Buffer (10x)
3 µl
EcoRI-HF
03 µl
SpeI-HF
180 µl
ddH2O
=210 µl
TOTAL
17.5 µl Mastermix + 2.5 µl Plasmid each
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V
1 kb Marker
P188 Alk.Phos.
P189 Alk.Phos.
P190 self lysis device
P191 self lysis device
P192 Nuclease
P193 Nuclease
P194 PIF6
P195 PIF6
P196 RBS
P197 RBS
successful
successful
successful
successful
successful
successful
successful
successful
successful
successful
[Expand] Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56
Investigator: Ingmar
Aim of the experiment: Picking of of E. coli XL1 blue transformed with the ligation products of F52+F53, F59+F58, F59+F54, F59+F55, F59+F57 and F59+F56
Procedure:
The picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x) or ampecillin (1000x).
2 colonies of each agar plate were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overday.
The ligation of F42 and F51 was not successful and has to be repeated. One reason for the failure could be secondary structures of the promoter. Therefore the new ligation batch should be heated to 95°C for a while before adding the T4 ligase.
[Expand] PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43)
Investigator: Jeff
Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65) and P194 (PIF6, O42 & O43).
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P192: O64 (NucA_fw); P194: O42 (PIF6_fw))
1 µl
10 µM Reverse Primer (P192: O65 (NucA_rv); P194: O43 (PIF6_rv))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
1:1000 dilution of plasmid DNA (P192; P194)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The gradient PCR program was performed after following scheme with following conditions:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
55 °C
60 s
68 °C
30 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
PCR product of P192 = F61; PCR product of 194 = F62
[Expand] QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P179 (Laccase)
Investigator: Jeff
Aim of the experiment: Removal of forbidden restiction site from P179 (Laccase).
Procedure: Quickchange - PCR
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid template (P179 - 1:10 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O32 (10 µM)
1 µl
1:10 dilution of O33 (10 µM)
18.5 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P179
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
4 min
4 °C
hold
After the PCR; 1 µl of Dpn1 was added and the reaction mixture was incubated at 37°C for one hour
[Expand] Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179
Investigator: Rosario, Andi, Jeff
Aim of the experiment: Analytical gelelectrophoresis of F61 (PCR product of P192, Thermonuclease), F62 (PCR product of P194, PIF6) and the DpnI digestion of the SDMIV product of P179.
Procedure:
4 µl of F61 or F62 or the DpnI digestion of the SDMIV product of P179 were mixed with 0.444 µl of DNA loading buffer (10x)
Analytical gelelectrophoresis was performed at 90 V for 1 h on a 1% agarose gel.
1 kbp DNA ladder
PCR product F61
PCR product F62
DpnI digestion of QCIV of P179
successful
successful
successful
[Expand] Ligation of F42+F51
Investigator: Ingmar
Aim of the experiment: Ligation of F42+F51.
Procedure:
Ligation batch for F42+F51:
volume
reagent
0.68 µl
F42 (73.5 ng/µl, 2070 bp)
15.64 µl
F51 (10.93 ng/µl, 1237 bp)
5.39 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The reaction batch was heated up to 95°C for 5 min before the enzyme and buffer were added.
The ligation was performed for two hours at room temperature.
Lid temperature = 37 °C
[Expand] Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10) and DpnI digestion of the SDMIV product of P179 (Laccase)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1 blue with F42+F51 (pActin in pSB1C3 RFC10 )and DpnI digestion of the SDMIV product of P179 (Laccase).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).
Aim of the experiment: Sequencing of the Biobrick MiniPreps
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM):
Label
Konz. ng/µl
Vol. BB µl
Vol H2O µl
P188
160.6
5
10
P189
123.6
7.5
7.5
P190
319.2
3
12
P191
127.0
10
5
P192
336.9
3
12
P193
230.6
4
11
P194
156.6
5
10
P195
197.4
4
11
P196
90.3
10
5
P197
230.2
4
11
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P188
alk.Phos.
FR01002327 (O3)
FR01002326 (O4)
P189
alk.Phos.
FR01002325 (O3)
FR01002320 (O4)
P190
self lysis device
FR01002319 (O3)
FR01002318 (O4)
P191
self lysis device
FR01002317 (O3)
FR01002316 (O4)
P192
Nuclease
FR01002315 (O3)
- (O4)
P193
Nuclease
FR01002314 (O3)
- (O4)
P194
PIF6
FR01002313 (O3)
- (O4)
P195
PIF6
FR01002312 (O3)
- (O4)
P196
RBS
FR01002324 (O3)
FR01002323 (O4)
P197
RBS
FR01002322 (O3)
FR01002321 (O4)
[Expand] Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII)
Investigator: Rosario, Andi, Florian, Johanna, Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of F61 (Thermonuclease), F62 (PIF6) , P165 (t35S), F31 (miniMCSII.
Procedure:
Batch for preparative digestion of F61 (Thermonuclease) with XbaI & AgeI:
volume
reagent
25 µl
PCR product F61
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of F62 (PIF6) with XbaI & AgeI:
volume
reagent
25 µl
PCR product F62
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P165 (t35S) with XbaI:
volume
reagent
20 µl
Plasmid DNA P165
4 µl
CutSmart Buffer
1 µl
XbaI (20 U/µl)
15 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of F31 (miniMCSII):
volume
reagent
10 µl
Oligohybridization F31
5 µl
CutSmart Buffer
1 µl
SpeI-HF (20 U/µl)
34 µl
ddH2O
=40 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches and 4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on an 2% agarose gel for preparative gelelectrophoresis (1% agarose gel for digestion of P165).
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
Digestion of F31 with SpeI-HF
Digestion of F61 with XbaI & AgeI-HF
Digestion of F62 with XbaI & AgeI-HF
Fragment length as expected; band was cut out
Fragment length as expected; band was cut out
Fragment length as expected; band was cut out
1 kbp DNA ladder
Digestion of P165 with XbaI
Fragment length as expected; band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Dephosphorylation of preparative digestion of P165 with FastAP
Investigator: Rosario, Jeff
Aim of the experiment: Dephosphorylation of preparative digestion of P165 with FastAP to prevent re-ligation.
Procedure:
After preparative digestion, gelelectrophoresis and gelextraction of P165, the digestion product was purified with QIAquick PCR Purification Kit, Qiagen.
Batch for the dephosphorylation of digestion product of P165
volume
reagent
30 µl
Digestion product of P165
4 µl
10X Fast AP buffer
2 µl
FastAP Thermosensitive Alkaline Phosphatase
4 µl
ddH2O
=40 µl
TOTAL
The reaction batch was spun briefly and incubated at 37 °C for 10 min.
The reaction was stopped by heating to 75 °C for 5 min.
The dephosphorylized digestion product of P165 product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting product was labelled as F63
[Expand] Ligation of F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51
Investigator: Jeff, Rosario
Aim of the experiment: Ligation of F53+F59, F58+F59, F55+F59, F54+F59, F56+F59, F57+F59, F52+F53, F42+F51.
Procedure:
Ligation batch for F59+F65 (Thermonuclease in pSB1C3 RFC25):
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
4.56 µl
F65 (14.1 ng/µl, 447 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
10.94 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F59+F66:
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
3.78 µl
F66 (11.3 ng/µl, 297 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
11.72 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F63+F64:
volume
reagent
12.05 µl
F63 (8.3 ng/µl, 2287 bp)
4.95 µl
F64 (4.3 ng/µl, 22 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
Ligation was performed at 16 °C overnight.
Friday, June 7th
[Expand] Picking of QC IV Laccase
Investigator: Johanna
Aim of the study: Picking of QC IV Laccase (concentrated) and QC IV Laccase (diluted)
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
3 colonies were picked for every Transformation product.
[Expand]Preparation of Knop-Medium for Moss
Investigator: Andi
Aim of the experiment: Preparation of Knop-Medium for Moss
Procedure:
All four prepared stocks have been: 25g/L KH2PO4; 25g/L KCl; 25g/L MgSO4 x 7 H2O; 100g/L Ca(NO3)2
Volume of prepared Medium: 2.6L
10 ml of each stock were converted into a 5L Erlenmeyer flask and filled up to 2.6L with destilled water (ELGA); 32.5mg FeSO4 x 7 H2O were added; the pH was set to 5.8 with KOH/NaOH
200ml of the prepared medium was filled into three 500ml Erlenmeyer flasks, so each Erlenmeyer flask contained 200 ml of the Knop-Medium; the rest (2L) was left in the 5L EM flask
All flasks have been autoclaved
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58
Investigator: Rosario
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F52+F53, F59+F54, F59+F55, F59+F56, F59+F57, F59+F58
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
F52+F53 I
P202
F52+F53 II
203
F59+F54 I
P204
F59+F54 II
P205
F59+F55 I
P206
F59+F55 II
P207
F59+F56 I
P208
F59+F56 II
P209
F59+F57 I
P210
F59+F57 II
P211
F59+F58 I
P212
F59+F58 II
P213
[Expand] Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)
Investigator: Florian
Aim of the experiment: Transformation of E. coli XL1 blue with F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of ligation product were added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of each tube were plated on agarose plates containing antibiotics (CamR).
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).
Aim of the experiment: Sequencing of Ig-Kappa (P204), Nanoluciferase (P206), SigP (P208), Transmembrand. (P210) and SpyCatcher (P212) Ligations in pSB1C3 RFC25
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Name
Barcode (O3)
P204
Ig-Kappa-SigP + pSB1C3 RFC25
FR01002304
P206
Nanoluciferase + pSB1C3 RFC25
FR01002303
P208
SERK-SigP + pSB1C3 RFC25
FR01002302
P210
Transmembrand + pSB1C3 RFC25
FR01002301
P212
SpyCatcher + pSB1C3 RFC25
FR01002300
[Expand] Analytical digestion and gelelectrophoresis of P202-P213
Investigator: Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P202-P213
Procedure:
Mastermix for analytical digestion of P202-P213 with XbaI+AgeI
volume
reagent
30 µl
CutSmart Buffer (10x)
3.75 µl
XbaI
3.75 µl
SpeI-HF
225 µl
ddH2O
=262.5 µl
TOTAL
17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P202-P203)
Incubation for 90 min at 37 °C.
Inaddition, P203 was digested for 4 and for 8 seconds in the microwave, a negative control was incubated at room temperature
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp ladder DNA ladder
Digestion of P202 with XbaI und AgeI (GFP + linker)
Digestion of P203 with XbaI und AgeI (GFP + linker)
Digestion of P204 with XbaI und AgeI
Digestion of P205 with XbaI und AgeI
Digestion of P206 with XbaI und AgeI
Digestion of P207 with XbaI und AgeI
Digestion of P208 with XbaI und AgeI
Digestion of P209 with XbaI und AgeI
Digestion of P210 with XbaI und AgeI
as expected
as expected
not as expected
not as expected
not as expected
not as expected
not as expected
not as expected
as expected (SERK-TMD)
1 kbp ladder DNA ladder
Digestion of P211 with XbaI und AgeI
Digestion of P212 with XbaI und AgeI
Digestion of P213 with XbaI und AgeI
Digestion of P203 with XbaI und AgeI (microwave 4 seconds)
Digestion of P203 with XbaI und AgeI (microwave 8 seconds)
Digestion of P203 with XbaI und AgeI (control)
as expected (SERK-TMD)
not as expected
as expected
interesting
interesting
interesting
Saturday, June 8th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase
Investigator: Rosario
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue QC IV Laccase
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
QC IV Laccase clone 1
P214
QC IV Laccase clone 2
P215
QC IV Laccase clone 3
P216
QC IV Laccase clone 4
P217
QC IV Laccase clone 5
P218
QC IV Laccase clone 6
P219
[Expand] Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)
Investigator: Rosario
Aim of the study: Picking of F59 + F65 (Thermonuclease in pSB1C3), F59 + F66 (PIF6 in pSB1C3) and F63 + F64 (MiniMCS + t35S in pSB1C3)
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
2 colonies were picked for every Transformation product.
[Expand] Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67)
Investigator: Jeff, Katrin
Aim of the experiment: Preparative digestion and gelelectrophoresis of F47 (pActin, fragment named F68) with XbaI/SpeI, P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI, P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
Procedure:
Batch for preparative digestion of F47 (pActin, fragment named F68) with XbaI/SpeI
volume
reagent
25 µl
PCR product F47
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
volume
reagent
20 µl
plasmid P202
5 µl
CutSmart Buffer
1 µl
SpeI-HF(20 U/µl)
1 µl
NgoMIV (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
volume
reagent
20 µl
Plasmid DNA P210
5 µl
CutSmart Buffer
1 µl
AgeI-HF (20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of F47 (pActin, fragment named F68) with XbaI/SpeI
Fragment length as expected; band was cut out
1 kbp DNA ladder
digestion of P202 (GFP+linker, fragment named F69) with NgoMIV/SpeI
Fragment length as expected; band was cut out
1 kbp DNA ladder
digestion of P210 (SERK-TMD, fragment named F67) with AgeI/SpeI
Fragment length as expected; band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentration determined with Nanodrop:
F68
pActin
10,4 ng/µl
F69
GFP+linker
13,0 ng/µl
F67
SERK-TMD
14,3 ng/µl
[Expand] Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
Investigator: Jeff, Katrin
Aim of the experiment: Preparative digestion and gelelectrophoresis of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI and P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI (backbones for ligation)
Procedure:
Batch for preparative digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI
volume
reagent
20 µl
Plasmid P7
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
volume
reagent
20 µl
plasmid P8
5 µl
CutSmart Buffer
1 µl
SpeI-HF(20 U/µl)
1 µl
NgoMIV (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
before gelelectrophoresis, P7 was dephosphorylated
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 0.5% agarose gel for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of P7 (PhyB in pSB1C3, fragment named F75) with XbaI/SpeI
digestion of P8 (PhyB in pSC1C3, fragment named F76) with NgoMIV/SpeI
Fragment lengths as expected; lower band was cut out
Fragment lengths as expected; lower band was cut out
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentration determined with Nanodrop:
F75
dephosphorylated Backbone (P7)
39,4 ng/µl
F76
dephosphorylated Backbone (P8)
52,0 ng/µl
[Expand] Dephosphorylation of F75
Investigator: Jeff, Katrin
Aim of the experiment: Dephosphorylation of cut vector pSB1C3 prevent re-ligation
Procedure:
the digestion product was purified with QIAquick PCR Purification Kit, Qiagen in order to change the buffer
Batch for the dephosphorylation of F75
volume
reagent
30 µl
Plasmid DNA F75 from digestion
3 µl
10X Fast AP buffer
1.3 µl
FastAP Thermosensitive Alkaline
Phosphatase
=34.3 µl
TOTAL
the reaction batch was spun briefly and incubated at 37 °C for 10 minutes
the reaction was stopped by heating to 75 °C for 5 minutes
[Expand] Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI
Investigator: Jeff, Katrin
Aim of the experiment: Preparative digestion and gelelectrophoresis of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI and P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeI
Procedure:
Batch for preparative digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI
volume
reagent
20 µl
Plasmid P7
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
volume
reagent
20 µl
Plasmid P7
5 µl
CutSmart Buffer
1 µl
XbaI(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Incubation for 3 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 2% agarose gel for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of P200 (gene synthesis 1, fragments named F70+F71) with XbaI/AgeI
digestion of P198 (gene synthesis 2, fragments named F72-F74) with XbaI/AgeII
Fragment lengths as expected; the two lower bands were cut out (upper:F70, lower: F71)
Fragment lengths as expected; all bands were cut out (upper: F72, middle: F73, lower: F74)
Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.
Concentrations measured with Nano-Drop
Fragment
Name
Length
Concentration
F70
Nanoluciferase
510 bp
9.2 ng/µl
F71
IgKappa-SigP
67 bp
3.1 ng/µl
F72
SpyCatcher
339 bp
10.8 ng/µl
F73
SERK-TMD
186 bp
8.6 ng/µl
F74
SERK-SigP
100 bp
4.8 ng/µl
[Expand] Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)
Investigator: Jeff, Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)
Procedure:
Mastermix for analytical digestion of P214-219 (Miniprep of Laccase QC IV), P179 (control before QC IV)
17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA (P214-219, P179)
Incubation for 90 min at 37 °C.
digestion for 4 x 10 seconds in the microwave (600 watt), with two minute breaks in between the microwaving
Analytical gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of P214 (Miniprep of Laccase QC IV clone 1) with NgoMVI/AgeI
digestion of P215 (Miniprep of Laccase QC IV clone 2) with NgoMVI/AgeI
digestion of P216 (Miniprep of Laccase QC IV clone 3) with NgoMVI/AgeI
digestion of P217 (Miniprep of Laccase QC IV clone 4) with NgoMVI/AgeI
digestion of P218 (Miniprep of Laccase QC IV clone 5) with NgoMVI/AgeI
digestion of P219 (Miniprep of Laccase QC IV clone 6) with NgoMVI/AgeI
digestion of P179 (Miniprep of ligation product Laccase QCIII clone 2) with NgoMVI/AgeI
as expected
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix)
Investigator: Jeff
Aim of the experiment: Ligation of F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into TMD-pSB1C3-Suffix).
Procedure:
volume
reagent
2.54 µl
F75 (39.4 ng/µl, ? bp)
8.30 µl
F69 (13.0 ng/µl, ? bp)
6.16 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
volume
reagent
7.00 µl
F67 (14.3 ng/µl, ? bp)
7.62 µl
F69 (13.0 ng/µl, ? bp)
2.38 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Incubation: 1 h 22°C
[Expand] Transformation of E. coli XL1 blue with F75+F69 (Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with F75+F69(Linker+GFP into pSB1C3) and F67+F69 (Linker+GFP into pSB1C3-TMD-Suffix)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).
Sunday, June 9th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64
Investigator: Katrin
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue of F59+F65 (Nuclease), F59+F66 (PIF6), F59+F54 (Ig-K), F59+F55 (Luciferase), F59+F56 (SERK-SigP), F59+F57 (SERK-TMD), F59+F58 (SpyCatcher), F42+F51 (pActin), F63+F64
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
F59+F65 clone 1
P220
F59+F65 clone 2
P221
F59+F66 clone 1
P222
F59+F66 clone 2
P223
F59+F56 clone 1
P224
F59+F56 clone 2
P225
F59+F55 clone 1
P226
F59+F55 clone 2
P227
F59+F57 clone 1
P228
F59+F57 clone 2
P229
F59+F58 clone 1
P230
F59+F58 clone 2
P231
F63+F64
P232
F42+F51
P233
F59+F54 clone 1
P234
F59+F54 clone 2
P235
[Expand] Analytical digestion and gelelectrophoresis of P220-P235 ((P220-231, P233-P235 with XbaI/SpeI, P232 with MfeI/Pst1)
Investigator: Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P220-P235 (Minipreps)
Procedure:
Mastermix for analytical digestion of P220-231, P233-P235 with XbaI, SpeI
volume
reagent
34 µl
CutSmart Buffer
4.25 µl
XbaI(20 U/µl)
4.25 µl
SpeI-HF (20 U/µl)
255 µl
ddH2O
=266.5 µl
TOTAL
17.5 µl of the mastermix was added to 2.5 µl of plasmid DNA
reaction batch for analytical digestion of P232 with MfeI/PstI
volume
reagent
2.5 µl
P232
2 µl
CutSmart Buffer
0.25 µl
MfeI(20 U/µl)
0.25 µl
PstI-HF(20 U/µl)
15 µl
ddH2O
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
for P224, P225, P228, P229, P230, P231, P234, P235,a 2% agarose gel was used, gelelectrophoresis was performed at 90 V for 45 min
1 kbp DNA ladder
100 bp DNA ladder
digestion of P224 (SERK-SigP, clone 1) with XbaI, SpeI
digestion of P225 (SERK-SigP, clone 2) with XbaI, SpeI
digestion of P228 (SERK TMD, clone 1) with XbaI, SpeI
digestion of P229 (SERK TMD, clone 2) with XbaI, SpeI
digestion of P230 (SpyCatcher, clone 1) with XbaI, SpeI
digestion of P231 (SpyCatcher, clone 2) with XbaI, SpeI
digestion of P234 (Ig-K-SigP, clone 1) with XbaI, SpeI
digestion of P235 (Ig-K-SigP, clone 2) with XbaI, SpeI
not as expected
not as expected
as expected
as expected
as expected
as expected
fragment not visible due to small size
not as expected
1 kbp DNA ladder
digestion of P220 (Thermonuclease, clone 1) with XbaI, SpeI
digestion of P221 (Thermonuclease, clone 2) with XbaI, SpeI
digestion of P222 (PIF6, clone 1) with XbaI, SpeI
digestion of P223 (PIF6, clone 2) with XbaI, SpeI
digestion of P226 (Luciferase, clone 1) with XbaI, SpeI
digestion of P227 (Luciferase, clone 2) with XbaI, SpeI
digestion of P232 (miniMCS) with MfeI/PstI
digestion of P233 (pActin) with XbaI, SpeI
not as expected
not as expected
as expected
as expected
as expected
as expected
not as expected
as expected
[Expand] Picking of F67+F69 (SERK-TMD+linker GFP)
Investigator: Katrin
Aim of the study: Picking of F67+F69 (SERK-TMD+linker GFP)
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (5 µl CamR and 5 ml LB-medium)
3 colonies were picked for every Transformation product.
There were no colonies on the plates for ligations F68+F75 (pActin, pSB1C3),
(probably due to dephosphorylation)
[Expand] Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI
Investigator: Katrin
Aim of the experiment: Preparative digestion and gelelectrophoresis of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI, P160 (NLS, fragment named F77) with AgeI/SpeI, P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI, P7 (PhyB, fragment named F78) with AgeI/SpeI
Procedure:
Batch for preparative digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI
volume
reagent
20 µl
P220
5 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P160 (NLS, fragment named F77) with AgeI/SpeI
volume
reagent
20 µl
plasmid P160
5 µl
CutSmart Buffer
1 µl
SpeI-HF(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
volume
reagent
20 µl
plasmid P40
5 µl
CutSmart Buffer
1 µl
SpeI-HF(20 U/µl)
1 µl
NgoMIV (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P7 (PhyB, fragment named F78) with AgeI/SpeI
volume
reagent
20 µl
plasmid P40
5 µl
CutSmart Buffer
1 µl
SpeI-HF(20 U/µl)
1 µl
AgeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches containing P220 and P40 after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
the vector backbones where only a few base pairs were cut out were purified via PCR-purification
Preparative gelelectrophoresis was performed at 90 V for 60 min.
digestion of P220 (thermonuclease, fragment corrupted) with NgoMIV/SpeI
1 kbp DNA ladder
digestion of P40 (GSAT linker, fragment named F79) with NgoMIV/SpeI
not as expected, band was discarded
as expected, lower band (108 bp) was cut out
the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)
Investigator: Katrin
Aim of the study: PCR Purification of vector backbones P7 (PhyB in pSB1C3, fragment F78), P160 (NLS in pSB1C3, fragment F77)
in order to get rid of the few base pairs that were cut out during digestion, the digestion batches were purified
with QIAquick PCR Purification Kit, Qiagen
the yiedls were 56.2 ng/µl for F77 and 277,6 ng/µl for F78
[Expand] Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)
Investigator: Katrin
Aim of the experiment: Ligation of F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)
Procedure:
Ligation batch for F78+F79(PhyB+linker)
volume
reagent
0.36 µl
F78 (277.6 ng/µl, 4807 bp)
1.77 µl
P79 (3.8 ng/µl, 108 bp)
14.87 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F59+F71 (pSB1C3+Ig-K)
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
3.12 µl
P71 (3.1 ng/µl, 67 bp)
12.38 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1 blue with F78+F79(PhyB+linker) and F59+F71 (pSB1C3+Ig-K) and negative controls F78 and F59
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).
[Expand] PCR of P192 (Thermonuclease, O64 & O65)
Investigator: Katrin
Aim of the experiment: PCR of P192 (Thermonuclease, O64 & O65)
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer O64 (NucA_fw)
1 µl
10 µM Reverse Primer O65 (NucA_rv)
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
1:1000 dilution of plasmid DNA (P192)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The gradient PCR program was performed after following scheme with following conditions:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
55 °C
60 s
68 °C
30 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
Week 8
Monday, June 10rd
[Expand] Analytical Gel of PCR of Thermonuclease (P192)
Investigator: Florian
Aim of the study: Analysis of PCR of Thermonuclease (P192); supposed letgth: 447 bp
Lane:
DNA ladder 1kb
P192 PCR product
DNA ladder 1kb
Result:
-
as expected
-
[Expand] PCR Purification of Thermonuclease (P192)
Investigator: Andi
Aim of the study: PCR Purification of Thermonuclease (P192)
After the PCR the batch was purified with QIAquick PCR Purification Kit, Qiagen
Eluation in 35 µl EB buffer -> F80
[Expand] Miniprep of F67+F69 Ligation (Linker+GFP in Suffix of TMD)
Investigator: Johanna
Aim of the study: Preparation of DNA of the F67+F69 Ligation from 09.06.13 (Linker+GFP in Suffix of TMD)
Procedure: according to MiniPrep Kit QIAGEN; measurement of concentration (NanoDrop)
Eluation in 35 µl EB buffer each
Plamsid
Concentration
P236
108.0 ng/µl
P237
116.0 ng/µl
P238
165.4 ng/µl
[Expand] Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)
Investigator: Johanna
Aim of the study: Picking of F78 + F79 (PhyB + Linker) and F59 + F71 (pSB1C3 + lgkappa)
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
3 colonies were picked for every Transformation product.
[Expand] Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)
Investigator: Johanna
Aim of the study: Sequencing of P150 (FluA), P233 (pActin), P213 (Spycatcher) and F67+F69 Ligation (Linker+GFP + TMD)
[Expand] PCR of BPul Laccase P219 + Analytical Gelelectrophoresis
Investigator: Andi, Johanna
Aim of the experiment: PCR of Bpul Laccase P219.
Procedure:
Operational sequence:
PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Reaction Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P219:O24 (Bpul_for))
1 µl
10 µM Reverse Primer (P143:O25 (Bpul_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P219)- 1:1000 dilution
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
55 °C
60 s
68 °C
90 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting fragment was called F82
Further on an analytif Gelelectrophoresis was performed to be sure that the expected fragment was amplified.
DNA ladder 1kb
P219 PCR product
As we can see, the fragment has the expected length of about 1600 BP.
[Expand] Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI
Investigator: Florian, Johanna
Aim of the experiment: Preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.
Procedure:
Batch for preparative digestion of F80 (PCR purification of Thermonuclease) with XbaI/AgeI.
volume
reagent
25 µl
F80
5 µl
CutSmart Buffer
1 µl
AgeI (20 U/µl)
1 µl
XbaI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation for 2.5 h at 37 °C.
The digested fragment was purified via PCR-purification (Qiagen-kit)
A yield of 26.2 ng/µl could be measured for F81
[Expand] Ligation of F59 + F81 (Thermonuclease in pSB1C3)
Investigator: Florian, Johanna
Aim of the experiment: Ligation of F59 + F81 (Thermonuclease in pSB1C3).
Procedure:
Ligation batch for F59 + F81 (Thermonuclease in pSB1C3)
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
2.45 µl
P81 (26.2 ng/µl, 447 bp)
13.55 µl
ddH2O
2 µl
T4 ligase buffer (10x)
0.5 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1 blue with F59+F81(Thermonuclease in pSB1C3) and the negative control F59.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Addition of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspensions were plated on plates containing antibiotics (CamR).
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on plates containing antibiotics (CamR).
[Expand] Preparative digestion and purification of F82 (PCR product of Bpul Laccase) with XbaI/AgeI-HF and PCR purification kit of Qiagen
Investigator: Andi, Johanna
Aim of the experiment: Preparative digestion and purification of F82 (Bpul Laccase) with XbaI+AgeI and PCR purification kit of Qiagen
Procedure:
Batch for preparative digestion of F82 with XbaI/AgeI
volume
reagent
25 µl
F82
5 µl
CutSmart Buffer
1 µl
AgeI-HF(20 U/µl)
1 µl
XbaI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation for 2.5 h at 37 °C.
the digested fragment was purified via PCR-purification
the resulting fragment was called F83 (F82 digested with XbaI+AgeI)
[Expand] Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)
Investigator: Andi, Johanna
Aim of the experiment: Ligation of F83(PCR product of Bpul Laccase digested with AgeI-HF + XbaI)and F59 (psB1C3 digested with AgeI-HF + XbaI)
Procedure:
Ligation batch for F83+F59 (psB1C3+Bpul Laccase)
volume
reagent
15.5 µl
F83 (9.2 ng/µl, 1600 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed over night at 16°C.
Tuesday, June 11th
[Expand] Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83
Investigator: Louise, Andi
Aim of the experiment: Transformation of E. coli XL1 blue with ligation products F59+F81 and F59+F83.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa)
Investigator: Louise, Johanna
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with F78+F79 (PhytochromB +Linker) and F59+F71 (IgKappa).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
F78+F79 (PhytochromB +Linker) clone 1
P239
F78+F79 (PhytochromB +Linker) clone 2
P240
F78+F79 (PhytochromB +Linker) clone 3
P241
F59+F71 (IgKappa) clone 4
P242
F59+F71 (IgKappa) clone 5
P243
F59+F71 (IgKappa) clone 6
P244
[Expand] Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI
Investigator: Louise, Rosario, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of P239-P241 (PhytochromB+Linker) and P242-P244 (IgKappa) with XbaI and AgeI
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
AgeI
0.25 µl
XbaI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P293-P244)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
For P242-P244 a 2% agarose gel was used and for P293-P241 a 0.5% agarose gel was used.
Lane:
Digestion of P242 (IgK) with XbaI & AgeI, clone 1
Digestion of P243 (IgK) with XbaI & AgeI, clone 2
Digestion of P244 (IgK) with XbaI & AgeI, clone 3
DNA ladder 100bp
Result:
as expected
as expected
as expected
-
Lane:
Digestion of P239 (PhyB+Linker) with XbaI & AgeI, clone 1
Digestion of P240 (PhyB+Linker) with XbaI & AgeI, clone 2
Digestion of P241 (PhyB+Linker) with XbaI & AgeI, clone 3
DNA ladder 1kb
Result:
failed
as expected
as expected
-
[Expand] Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI
Investigator: Johanna, Rosario, Flo
Aim of the experiment: Preparative digestion and gelelectrophoresis of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI, P157 (EreB, fragment named F86) with NgoMIV/SpeI, P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI, P213 (Spycatcher, fragment named F88) with AgeI/SpeI and P170 (Spytag, fragment named F89) with NgoMIV/SpeI
Procedure:
Batch for preparative digestion of P204 (IgKappa-SigP, fragment named F90) with AgeI/SpeI
volume
reagent
20 µl
P204
5 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P157 (EreB, fragment named F86) with NgoMIV/SpeI
volume
reagent
20 µl
P157
5 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P208 (NanoLuc, fragment named F87) with NgoMIV/SpeI
volume
reagent
20 µl
P208
5 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P213 (Spycatcher, fragment named F88) with NgoMIV/SpeI
volume
reagent
20 µl
P213
5 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Batch for preparative digestion of P170 (Spytag, frament named F89) with NgoMIV/SpeI
volume
reagent
20 µl
P170
5 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Incubation for 2.5 h at 37 °C.
5 µl of DNA loading buffer (10x) were added to the 50 µl reaction batches after digestion and were loaded on a 1% agarose gel (P204, P157, P208, P213) and 2 % agarose gel (P170) for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of P204 (IgKappa-SigP) with AgeI/SpeI
100 bp DNA ladder (Generuler)
digestion of P157 (EreB) with NgoMIV/SpeI
as expected, lower band was cut out
as expected, lower band was cut out
1 kbp DNA ladder
digestion of P208 (Nanoluc) with NgoMIV/SpeI
100 bp DNA ladder (Generuler)
digestion of P213 (Spycatcher) with NgoMIV/SpeI
as expected, lower band was cut out
as expected, lower band was cut out
100 bp DNA ladder (Generuler)
digestion of P170 (Spytag) with NgoMIV/SpeI
as expected, lower band was cut out
the band was extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)
Investigator: Johanna
Aim of the experiment: Ligation of F90+F86 (IgKappa-SigP + EreB), F90+F87 (IgKappa-SigP + Nanoluciferase), F90+F88 (IgKappa-SigP + SpyCatcher), F90+F89 (IgKappa-SigP + SpyTag) and negativ control of F90 (IgKappa-SigP)
Procedure:
Ligation batch for F90+F86 (IgKappa-SigP + EreB)
volume
reagent
5.7 µl
F86 (31.0 ng/µl, 1254 bp)
6.5 µl
F90 (15.3 ng/µl, 2144 bp)
4.8 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F90+F87 (IgKappa-SigP + Nanoluciferase)
volume
reagent
7.0 µl
F87 (10.2 ng/µl, 510 bp)
6.5 µl
F90 (15.3 ng/µl, 2144 bp)
3.5 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F90+F88 (IgKappa-SigP + SpyCatcher)
volume
reagent
6.3 µl
F88 (8.7 ng/µl, 339 bp)
6.5 µl
F90 (15.3 ng/µl, 2144 bp)
4.2 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F90+F89 (IgKappa-SigP + SpyTag)
volume
reagent
10.5 µl
F89 (8.7 ng/µl, 39 bp)
6.5 µl
F90 (15.3 ng/µl, 2144 bp)
0.0 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control of F 90 (IgKappa-SigP) was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.
[Expand] PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!
Investigator: Andi
Aim of the experiment: PCR of pActin (P143) and Thermonuclease (P192) to find the best conditions for PCR + Measurement via Nanodrop + Analytical Gelelectrophoresis to get to know about it!
Procedure:
Operational sequence:
The PCR for P143 (pActin) was performed three times under different conditions
First batch - PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Reaction Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P143:O62 (pAct_for))
1 µl
10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P143)- 1:1000 dilution
35.75 µL
ddH2O Water
=50 µL
TOTAL
Second batch - PCR reaction mixture
volume
reagent
10 µl
5x OneTaq GC Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P143:O62 (pAct_for))
1 µl
10 µM Reverse Primer (P143:O63 (pAct_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P143)- 1:1000 dilution
35.75 µL
ddH2O Water
=50 µL
TOTAL
Third batch - PCR reaction mixture
volume
reagent
10 µl
5x Herculase Fusion Polymerase reaction buffer
2.5 µl
10 mM dNTPs
1.25 µl
10 µM Forward Primer (P143:O62 (Bpul_for))
1.25 µl
10 µM Reverse Primer (P143:O63 (Bpul_rev))
1 µL
Herculase II Fusion Polymerase
2 µl
Plasmid DNA (P143)- 1:1000 dilution
0.5 µl
DMSO
35.75 µL
ddH2O Water
=50 µL
TOTAL
The content of all batches has been mixed with a pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
51 °C
60 s
68 °C
90 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
The PCR for P192(Thermonuclease) was performed three times under different conditions
First batch - PCR reaction mixture
volume
reagent
10 µl
5x OneTaq Reaction Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl
10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P192)- 1:1000 dilution
35.75 µL
ddH2O Water
=50 µL
TOTAL
Second batch - PCR reaction mixture
volume
reagent
10 µl
5x OneTaq GC Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (P192:O64 (NucA_fw))
1 µl
10 µM Reverse Primer (P192:O65(NucA_rev))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P192)- 1:1000 dilution
35.75 µL
ddH2O Water
=50 µL
TOTAL
Third batch - PCR reaction mixture
volume
reagent
10 µl
5x Herculase II DNA Fusion Polymerase reaction Buffer
2.5 µl
10 mM dNTPs
1.25 µl
10 µM Forward Primer (P192:O64 (NucA_fw))
1.25 µl
10 µM Reverse Primer (P192:O65(NucA_rev))
1 µL
Herculase II Fusion DNA Polymerase
3 µl
Plasmid DNA (P192)- 1:1000 dilution
0.5 µL
DMSO
30.5 µL
ddH2O Water
=50 µL
TOTAL
The PCR program of the Thermonuclease (P192) was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
54 °C
60 s
68 °C
40 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
Mix the content of the batches with pipette
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The concentrations of all PCR products have been measured
P143 - Batch #1 - PCR product - 5.9 ng/µl
P143 - Batch #2 - PCR product - 40.5 ng/µl
P143 - Batch #3 - PCR product - 12.4 ng/µl
P192 - Batch #1 - PCR product - 39.7 ng/µl
P192 - Batch #2 - PCR product - 23.1 ng/µl
P192 - Batch #3 - PCR product - 82.5 ng/µl
As we can see, the highest concentration of PCR product seems to be in P143 - Batch #2 and P192 - Batch #3.
Further on an analytic Gelelectrophoresis was performed to be sure that the expected fragment was amplified.
DNA ladder 1kb
P143 - Batch #1 - PCR product
P143 - Batch #2 - PCR product
P143 - Batch #3 - PCR product
P192 - Batch #1 - PCR product
P192 - Batch #2 - PCR product
P192 - Batch #3 - PCR product
As we can see, the fragment has the expected length of about 1600 BP referring to the PCR product of P143 and about XXX BP referring to the product of P192.
Further on the analytical gel confirms the output of the nano drop.
[Expand] Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI
Investigator: Andi
Aim of the experiment: Preparative digestion of PCR reaction batch #2 of P143 (pActin) XbaI/PstI and PCR reaction batch #3 of P192 with XbaI/AgeI
Procedure:
Batch for preparative digestion of PCR reaction batch #2 of P143 (pActin) with XbaI/PstI.
volume
reagent
24 µl
PCR reaction batch #2 of P143
5 µl
CutSmart Buffer
1 µl
PstI #1 (20 U/µl)
1 µl
PstI #2 (20 U/µl)
1 µl
XbaI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
I used 1 µl of both PstI batches because one seems to be broken
Incubation for 2.5 h at 37 °C.
The digested fragment was purified via PCR-purification (Qiagen-kit)
A yield of 16.9 ng/µl could be measured for the resulting F84
Batch for preparative digestion of PCR reaction batch #3 of P192 (Thermonuclease) with XbaI/AgeI-HF.
volume
reagent
24 µl
PCR reaction batch #2 of P143
5 µl
CutSmart Buffer
1 µl
AgeI-HF (20 U/µl)
1 µl
XbaI (20 U/µl)
19 µl
ddH2O
=50 µl
TOTAL
Incubation for 2.5 h at 37 °C.
The digested fragment was purified via PCR-purification (Qiagen-kit)
A yield of 46.2 ng/µl could be measured for the resulting F85
[Expand] Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) and F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI and F59 (psB1C3 digested with AgeI-HF + XbaI)
Investigator: Andi
Aim of the experiment: Ligation of F84(Digestion of PCR product (GC rich buffer) of P143 (pActin) with PstI/XbaI) with F42 (psB1C3 digested with PstI/XbaI) and Ligation of F85(Digestion of PCR product (Herculase II) of P192 (Thermonuclease) with XbaI/AgeI-HF) with F59 (psB1C3 digested with AgeI-HF/XbaI)
Procedure:
Ligation batch for F84+F42 (psB1C3+pActin)
volume
reagent
12.6 µl
F84 (16.9 ng/µl, 1500 bp)
1.4 µl
F42 (73.5 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
4 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F85+F59 (psB1C3+Thermonuclease)
volume
reagent
1.6 µl
F85 (46.2 ng/µl, 500 bp)
1.5 µl
F59 (66.7 ng/µl, 2100 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
13.9 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared for both batches. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed over night at 16°C.
[Expand] Transformation of E. coli XL1 blue with plasmids P157, P208 and P213
Investigator: Florian
Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P157, P208 and P213.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
Wednesday, June 12th
[Expand] Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chlorampenicol plates.
[Expand] Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI
Investigator: Louise, Florian
Aim of the experiment: Preparative digestion and gelelectrophoresis of P240 (PhyB + Linker) with AgeI/SpeI, P244 (IgKappa) with AgeI/SpeI, P197 (XylE) with NgoMIV/SpeI, P150 (FluA) with NgoMIV/SpeI, P126 (N-TEV) with AgeI/SpeI, P127 (C-TEV) with NgoMIV/SpeI, P168 (PIF3) with NgoMIV/SpeI and P222 (PIF6) with NgoMIV/SpeI
Procedure:
Batch for preparative digestion of P240 (PhyB + Linker) with AgeI/SpeI
volume
reagent
20 µl
P240
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P244 (IgKappa) with AgeI/SpeI
volume
reagent
20 µl
P244
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P197 (XylE) with NgoMIV/SpeI
volume
reagent
20 µl
P197
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P150 (FluA) with NgoMIV/SpeI
volume
reagent
20 µl
P150
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P126 (N-TEV) with AgeI/SpeI
volume
reagent
20 µl
P126
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P127 (C-TEV) with NgoMIV/SpeI
volume
reagent
20 µl
P127
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P168 (PIF3) with NgoMIV/SpeI
volume
reagent
20 µl
P168
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P222 (PIF6) with NgoMIV/SpeI
volume
reagent
20 µl
P222
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis
Preparative gelelectrophoresis was performed at 90 V for 60 min.
1 kbp DNA ladder
digestion of P126 (N-TEV) with AgeI/SpeI
digestion of P127 (C-TEV) with NgoMIV/SpeI
digestion of P150 (FluA) with NgoMIV/SpeI
as expected, upper band was cut out
as expected, lower band was cut out
as expected, lower band was cut out
1 kbp DNA ladder
digestion of P168 (PIF3) with NgoMIV/SpeI
digestion of P197 (XylE) with NgoMIV/SpeI
digestion of P222 (PIF6) with NgoMIV/SpeI
as expected, lower band was cut out
as expected, lower band was cut out
as expected, lower band was cut out
100 bp DNA ladder (Generuler)
digestion of P244 (IgKappa) with AgeI/SpeI
digestion of P240 (PhyB + Linker) with AgeI/SpeI
1 kbp DNA ladder
as expected, upper band was cut out
as expected, upper band was cut out
the DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN.
the fragments received the following names:
P126 (N-TEV)
F93
P127 (C-TEV)
F94
P150 (FluA)
F95
P168 (PIF3)
F96
P197 (XylE)
F97
P222 (PIF6)
F98
P240 (PhyB + Linker)
F91
P244 (IgKappa)
F92
[Expand] Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa)
Investigator: Florian
Aim of the experiment: Transformation of E. coli XL1 blue with plasmids P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension were plated on chloramphenicol plates.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated again on new chlorampenicol plates.
[Expand] Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3)
Investigator: Jeff
Aim of the study: Picking of P157, P208, P213, F59 + F81 (Thermonuclease in pSB1C3) and F59 + F83 (Laccase in pSB1C3).
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
2 colonies were picked for every ligation product and 1 colony was picked for every transformation product.
[Expand] Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls
Investigator: Jeff, Flo, Louise, Christopher
Aim of the experiment: Ligation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls
Procedure:
Ligation batch for F92+F89 :
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2153 bp)
16.14 µl
F89 (6.8 ng/µl, 39 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
0.0 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F92+F87 :
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2153 bp)
7.0 µl
F87 (10.2 ng/µl, 510 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
9.14 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F92+F86:
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2153 bp)
5.7 µl
F86 (31.0 ng/µl, 1260 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
10.44 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F92+F88 :
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2153 bp)
5.4 µl
F88 (8.7 ng/µl, 339 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
10.74 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F92+F97:
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2153 bp)
6.6 µl
F97 (19.4 ng/µl, 918 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
9.5 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F90+F97:
volume
reagent
6.5 µl
F90 (15.3 ng/µl, 2144 bp)
6.6 µl
F97 (19.4 ng/µl, 918 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
3.9 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F91+F94:
volume
reagent
0.63 µl
F91 (159 ng/µl, 4129 bp)
4.6 µl
F94 (5.6 ng/µl, 354 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
11.77 µl
ddH2O
=20 µl
TOTAL
Ligation batch for F93+F79:
volume
reagent
3.6 µl
F93 (27.4 ng/µl, 2440 bp)
3.5 µl
F79 (3.8 ng/µl, 108 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
9.9 µl
ddH2O
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed at room temperature for 1 hour
[Expand] Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls
Investigator: Jeff, Florian
Aim of the experiment: Transformation of E. coli XL1 blue with F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79 and their negative controls.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.
Thursday, June 13th
[Expand] QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P250, P251, P219 (Laccase), P163 (NPT-Cassette)
Investigator: Andi
Aim of the experiment: Removal of forbidden restriction site from P251 (Laccase) and P163 (NPT-Cassette).
Procedure: Quickchange - PCR
Reaction batch for P219 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P251 - 1:20 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O74 (10 µM)
1 µl
1:10 dilution of O75 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P163 (NPT-Cassette)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P163 - 1:60 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O70 (10 µM)
1 µl
1:10 dilution of O71 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P250 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P250 - 1:20 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O74 (10 µM)
1 µl
1:10 dilution of O75 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P217 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P217 - 1:100 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O74 (10 µM)
1 µl
1:10 dilution of O75 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
4 min
4 °C
hold
After the PCR, the Quickchange product was stored in the fridge.
[Expand] PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8) and analytical gel electrophoresis
Investigator: Louise, Johanna, Christopher
Aim of the experiment: PCR of AlcR (P61, pAutoRex8) and IRES (P61, pAutoRex8).
Procedure:
Operational sequence:
PCR reaction mixture for AlcR
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (O78 (AlcR_fw))
1 µl
10 µM Reverse Primer (O79 (AlcR_rv))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P61)
35.75 µL
ddH2O Water
=50 µL
TOTAL
PCR reaction mixture for IRES
volume
reagent
10 µl
5x OneTaq Standard Reaction Buffer
1 µl
10 mM dNTPs
1 µl
10 µM Forward Primer (O68 (IRES_fw))
1 µl
10 µM Reverse Primer (O69 (IRES_rv))
0.25 µL
OneTaq Hot Start DNA Polymerase (Finally: 1.25 units/50 µL)
1 µl
Plasmid DNA (P61)
35.75 µL
ddH2O Water
=50 µL
TOTAL
Mix with pipette
The gradient PCR program was performed after following scheme with following conditions (Tm=56 °C; ΔG=4 °C; AlcR in row 11(=60 °C); IRES in row 1 (=52.0 °C)):
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
Tm=56 °C; ΔG=4 °C
60 s
68 °C
160 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Resulting products was labeled as F99 (AlcR) and F100 (IRES).
[Expand] Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES)
Investigator: Louise, Christopher
Aim of the experiment: Analytical gelelectrophoresis of F99 (AlcR) and F100 (IRES).
Procedure:
4 µl of F99 and F100 were mixed with 0.44 µl DNA loading buffer (10x)
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
1 kbp DNA ladder
F100
F99
as expected
as expected
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)
Investigator: Rosario
Aim of the experiment:Miniprep of overnight cultures of transformated E. coli XL1 blue with P157, P208, P213, F59+F81(Thermonuclease), F59+F83 (Laccase)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
P157
P245
P208
P246
P213
P247
F59+F81 I
P248
F59+F81 II
P249
F59+F83 I
P250
F59+F83 II
P251
[Expand] Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (Laccase) with EcoRI and SpeI
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P248-P249 (Thermonuclease) and P250-251 (LAccase) with EcoRI and SpeI
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
SpeI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P248-P251)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
For P248-P251 a 1% agarose gel was used.
Lane:
DNA marker 1kb
Digestion of P248 with EcoRI & SpeI
Digestion of P249 with EcoRI & SpeI
Digestion of P250 with EcoRI & SpeI
empty
Digestion of P251 with EcoRI & SpeI
Result:
-
as expected
as expected
no insert (Laccase)
-
no insert (Laccase)
[Expand] Transformation of E. coli XL1 blue with P204
Investigator: Johanna
Aim of the experiment: Transformation of E. coli XL1 blue with P204
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.
[Expand] Sequencing of P61 (pSH21/pAutoRex8)
Investigator: Rosario, Katrin
Aim of the study: Sequencing of P61 (pSH21/pAutoRex8)
Aim of the study: Picking of F92+F89, F92+F87, F92+F86, F92+F88, F92+F97, F90+F97, F91+F94, F93+F79, P126 (N-TEV), P127 (C-TEV), P222 (PIF6), P240 (PhyB+Linker) and P244 (IgKappa), F42+F84, F90+F86, F90+F87, F90+F88, F90+F89, F59+F85
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR and 4 ml LB-medium)
There were no colonies on plates F42+F84 and xy
There were a lot of colonies on NK 90 but there were significantly more colonies on the plates with ligation products
2 colonies were picked for every ligation product.
Friday, June 14th
[Expand] Picking of transformed Plasmid E. coli XL1 blue with P204, F92+F86 and F42+F84
Investigator: Johanna
Aim of the study: Picking of P204, F92+F86 and F42+F84
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl CamR, 4 ml LB-medium)
3 colonies were picked for every transformation product.
[Expand] Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue
Investigator: Rosario, Johanna, Andi
Aim of the experiment:Mass of Minipreps of overnight cultures of transformated E. coli XL1 blue.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
F59+F85
P252
F59+F85
P253
F90+F86
P254
F90+F86
P255
F90+F87
P256
F90+F87
P257
F90+F88
P258
F90+F88
P259
F90+F89
P260
F90+F89
P261
F90+F97
P262
F90+F97
P263
F91+F94
P264
F91+F94
P265
F92+F87
P266
F92+F87
P267
F92+F97
P268
F92+F97
P269
F92+F88
P270
F92+F88
P271
F92+F89
P272
F92+F89
P273
F93+F79
P274
F93+F79
P275
P222
P276
P222
P277
P240
P278
P240
P279
P126
P280
P126
P281
P244
P282
P244
P283
P127
P284
P127
P285
[Expand] Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with P250, P251, P219 (Laccase), P163 (NPT-Cassette)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
Centrifugation for 1 min at 13000 rpm and the supernatant was dicarded.
The pellets were resuspended in 100 µl of LB-medium and these concentrated cell suspensions were plated on new chloramphenicol plates.
[Expand] Sequencing of P276 (Retrafo of P222, fw), P282 (Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv)
Investigators: Louise
Aim of the experiment: Sequencing of P276(Retrafo of P222, fw), P282(Retrafo of P244, fw), P278 (Retrafo of P240, fw+rv) and P217 (fw+rv).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P276
PIF6
FR02305830 (O3)
-
P282
IgKappa-SigP in pSB1C3
FR02305831 (O3)
P278
PhyB-36AALinker in pSB1C3
FR02305832 (O3)
FR02305833 (O4)
P217
Laccase QCIV
FR02305826 (O3)
FR02305827 (O4)
[Expand] Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI
Investigator: Rosario, Johanna, Louise
Aim of the experiment: Analytical digestion and gelelectrophoresis of P252-P275 with EcoRI and SpeI
Procedure:
Batches for every Plasmid of P252-P275 contained
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
SpeI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P252-P275)
=20 µl
TOTAL
The batches for the Quickchangeproduct were prepared with 6 µl of Quickchangeproduct and 2 µl DNA loading buffer.
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
For P252-P275 a 1% agarose gel was used.
Lane:
DNA marker 1kb
Digestion of P252 with EcoRI & SpeI
Digestion of P253 with EcoRI & SpeI
Digestion of P254 with EcoRI & SpeI
Digestion of P255 with EcoRI & SpeI
Digestion of P256 with EcoRI & SpeI
Digestion of P257 with EcoRI & SpeI
Digestion of P258 with EcoRI & SpeI
Result:
-
corrupt
as expected
corrupt
corrupt
corrupt
as expected
corrupt
Lane:
DNA marker 1kb
Digestion of P259 with EcoRI & SpeI
Digestion of P260 with EcoRI & SpeI
Digestion of P261 with EcoRI & SpeI
Digestion of P262 with EcoRI & SpeI
Digestion of P263 with EcoRI & SpeI
Digestion of P264 with EcoRI & SpeI
Digestion of P265 with EcoRI & SpeI
Result:
-
as expected
as expected (sequencing necessary)
as expected (sequencing necessary)
bad miniprep
as expected
not sure (has to be repeated with control P278)
not sure (has to be repeated with control P278)
Lane:
DNA marker 1kb
Digestion of P266 with EcoRI & SpeI
Digestion of P267 with EcoRI & SpeI
Digestion of P268 with EcoRI & SpeI
Digestion of P269 with EcoRI & SpeI
Digestion of P270 with EcoRI & SpeI
Digestion of P271 with EcoRI & SpeI
Digestion of P272 with EcoRI & SpeI
Result:
-
as expected
as expected
as expected
as expected
as expected
as expected
as expected (has to be sequenced)
Lane:
DNA marker 1kb
Digestion of P273 with EcoRI & SpeI
Digestion of P274 with EcoRI & SpeI
Digestion of P275 with EcoRI & SpeI
Digestion of QC I product of P251 with DpnI
Digestion of QC I product of P163I with DpnI
Digestion of QC V product of P163II with DpnI
Digestion of QC V product of P217 with DpnI
Result:
-
as expected (has to be sequenced)
as expected (has to be sequnced)
as expected (has to be sequnced)
corrupt, no QC product
corrupt, no QC product
corrupt, no QC product
as expected, QC product
[Expand] Preparative Digestion of IRES (F100) and AlcR (F99) with EcoRI and SpeI
volume
reagent
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI
1 µl
SpeI
9 µl
ddH2O
25 µl
Fragment DNA (F99 or F100)
=40 µl
TOTAL
Incubation for 150 min at 37 °C
Purification by QIAGEN PCR Purification Kit
new Names: F99 -> F101 and F100 -> F102
[Expand] Oligohybridization of Strep-TEV (O90 + O91)
Investigator: Rosario
Aim of the experiment: Oligohybridization of Strep-TEV (O90 + O91)
Procedure:
25 µL of 100 pmol/µl of Strep-TEV_fw and 25 µL of 100 pmol/µl Strep-TEV_rev
Heating up to 95 °C for 5 min
Cooling at RT in a styropor box overnight
Saturday, June 15th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3)
Investigator: Jeff
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P204 (IgKappa-SigP in pSB1C3), F92+F82 (IgKappa-SigP_EreB in pSB1C3).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
Concentration in ng/µl
ReTraFo of P204 (IgKappa-SigP in pSB1C3)
P286
105.7
ReTraFo of P204 (IgKappa-SigP in pSB1C3)
P287
303.6
ReTraFo of P204 (IgKappa-SigP in pSB1C3)
P288
268.1
F92+F86 (IgKappa-SigP_EreB in pSB1C3)
P289
121.2
F92+F86 (IgKappa-SigP_EreB in pSB1C3)
P290
402.3
F92+F86 (IgKappa-SigP_EreB in pSB1C3)
P291
405.2
[Expand] Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus)
Investigator: Jeff
Aim of the experiment: Ligation of F41+F101 (AlcR) and F41+F102 (IRES of poliovirus).
Procedure:
Ligation batch for F41+F101 (AlcR), 50 ng vector used, 1:1:
volume
reagent
0.56 µl
F41 (89.9 ng/µl, 2086 bp)
5.23 µl
F101 (11.3 ng/µl, 2466 bp)
11.21 µl
ddH2O
2 µl
T4 ligase buffer (10x)
0.5 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F41+F102 (IRES of poliovirus), 100 ng vector used, 3:1:
volume
reagent
1.11 µl
F41 (89.9 ng/µl, 2086 bp)
7.92 µl
F102 (11.4 ng/µl, 628 bp)
7.97 µl
ddH2O
2 µl
T4 ligase buffer (10x)
0.5 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
The ligation was performed for 1 hour at room temperature.
[Expand] Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P289-P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI.
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI-HF
0.25 µl
SpeI-HF
15 µl
ddH2O
2.5 µl
Plasmid DNA (P289-P291, P245)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
Lane:
DNA ladder 100bp
Digestion of P289 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 1
Digestion of P290 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 2
Digestion of P291 (IgKappa-SigP_EreB in pSB1C3) with EcoRI & SpeI, clone 3
Digestion of P245 (EreB in pSB1C3) with EcoRI & SpeI
Result:
as expected
as expected
as expected
as expected (control)
[Expand] Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with F41+F101 (AlcR in pSB1C3) and F41+F102 (IRES in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of ligation product was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of the cell suspension was plated on one chloramphenicol plate.
The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Sunday, June 16th
[Expand] QuikChange mutagenesis to remove forbidden restriction sites - SDMIV - of P217 (Laccase), P163 (NPT-Cassette)
Investigator: Andi
Aim of the experiment: Removal of forbidden restriction site from P217 (Laccase) and P163 (NPT-Cassette).
Procedure: Quikchange - PCR
Reaction batch for P217 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P217 - 1:100 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O74 (10 µM)
1 µl
1:10 dilution of O75 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P163 (NPT-Cassette)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P163 - 1:50 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O70 (10 µM)
1 µl
1:10 dilution of O71 (10 µM)
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
5 min
4 °C
hold
After the PCR, the Quickchange product was stored in the fridge.
[Expand] Picking of transformed Plasmid E. coli XL1 blue with QC of P217, P163, F41+F101 and F41+F102
Investigator: Andi, Jeff
Aim of the study: Picking of QC of P217, P163, F41+F101 and F41+F102
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
1 colonies were picked for every transformation product.
[Expand] Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV)
Investigator: Andi, Jeff
Aim of the experiment: Ligation of F59+F74 (SERK-SigP) and F59+F103 (Strep-TEV).
Procedure:
Ligation batch for F59+F74 (SERK-SigP), 100 ng vector used, insert:vector = 3:1
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
3 µl
F74 (4.8 ng/µl, 100 bp)
12.5 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F59+F103 (Strep-TEV), 100 ng vector used, insert:vector = 3:1
volume
reagent
1.5 µl
F59 (66.7 ng/µl, 2086 bp)
15.5 µl
F103 (504.9 ng/µl, 54 bp)
7.97 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)
Investigator: Andi, Jeff
Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013) and P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of ligation product/ 2 µl of plasmid (P7) was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
Week 9
Monday, June 17th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES)
Investigator: Rosario
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P163 (NPT-casette), QC V of P217 (laccase), ligation products F41+F101 (AlcR) and F41+F102 (IRES)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
QC I of P163
P292
QC V of P217
P293
F41+F101 I
P294
F41+F101 II
P295
F41+F101 III
P296
F41+F102 I
P297
F41+F102 II
P298
F41+F102 III
P299
[Expand] Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)
Investigator: Rosario
Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013), P7 (Retrafo of PhyB) and ligation products F59+F74 (SERK-SigP in pSB1C3) and F59+F103 (Strep-TEV in pSB1C3)
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
1 colony was picked for every transformation product.
[Expand] Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI
Investigator: Johanna, Flo
Aim of the experiment: Preparative digestion and gelelectrophoresis of P264 (PhyBlinker+cTev) with AgeI/SpeI, P299 (IRES) with NgoMIV/SpeI, P274 (nTevlinker)with AgeI/SpeI, P111 (TEV) with AgeI/SpeI and P248 (Nuclease) with NgoMIV/SpeI
Procedure:
Batch for preparative digestion of P264 (PhyBlinker+cTev) with AgeI/SpeI
volume
reagent
20 µl
P264
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P299 (IRES) with NgoMIV/SpeI
volume
reagent
20 µl
P299
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P274 (nTevlinker)with AgeI/SpeI
volume
reagent
20 µl
P274
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P111 (TEV) with AgeI/SpeI
volume
reagent
20 µl
P111
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P248 (Nuclease) with NgoMIV/SpeI
volume
reagent
20 µl
P248
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and were loaded on a 1% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane:
Digestion of P111 with AgeI & SpeI
1 kbp DNA ladder
Digestion of P248 with NgoMIV & SpeI
Digestion of P264 with AgeI & SpeI
Result:
as expected
as expected
as expected
Lane:
Digestion of P274 with AgeI & SpeI
1 kbp DNA ladder
Digestion of P299 with NgoMIV & SpeI
Result:
low concentration, discarded
not digested, discarded
The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN
[Expand] Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P293-P299 with EcoRI & SpeI and P292 with EcoRI & PstI
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI-HF
0.25 µl
SpeI-HF (PstI-HF for P292)
15 µl
ddH2O
2.5 µl
Plasmid DNA (P292-P299)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
Lane:
1 kbp DNA ladder
Digestion of P292 (QC I P163 (NPT-casette)) with EcoRI & PstI
Digestion of P293 (QC V P217 (Laccase)) with EcoRI & SpeI
Digestion of P294 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 1
Digestion of P295 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 2
Digestion of P296 (F41+F101 (AlcR)) with EcoRI & SpeI, clone 3
Digestion of P297 (F41+F102 (IRES)) with EcoRI & SpeI, clone 1
Digestion of P298 (F41+F102 (IRES)) with EcoRI & SpeI, clone 2
Digestion of P299 (F41+F102 (IRES)) with EcoRI & SpeI, clone 3
1 kbp DNA ladder
Result:
corrupt
corrupt
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] DpnI digestion of P163QCI and P217QCV
Investigator: Flo, Jeff
Aim of the experiment: DpnI digestion of P163QCI and P217QCV.
Procedure:
1 µl of DpnI was added to the QuikChange products of P163QCI and P217QCV.
The mixtures were incubated for 1 h at 37 °C; afterwards they were ready for transformation.
[Expand] Analytical gelelectrophoresis of DpnI digestion of P163QCI and P217QCV
Investigator: Jeff, Flo
Aim of the experiment: Analysis of the DpnI digestion of P163QCI and P217QCV
Procedure:
4.5 µl of the QuikChange products of P163 and P217, named P163QCI and P217QCV were mixed together with 0.5 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 30 min.
Lane:
1 kbp DNA ladder
DpnI digestion of P163QCI.1
DpnI digestion of P163QCI.2
DpnI digestion of P217QCV.1
DpnI digestion of P217QCV.2
Result:
empty
empty
empty
empty
[Expand] Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease)
Investigator: Jeff, Flo
Aim of the experiment: Ligation of F77+F105 (NLS in pSB1C3 + mature thermonuclease).
Procedure:
Ligation batch for F77+F105 (NLS in pSB1C3 + mature thermonuclease), 100 ng vector used, insert:vector = 3:1
volume
reagent
1.78 µl
F77 (56.2 ng/µl, 2143 bp)
5.22 µl
F105 (12 ng/µl, 447 bp)
10 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed overnight at 16 °C.
[Expand] Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations
Investigator: Flo
Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange product/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv)
Investigators: Rosario
Aim of the experiment: Sequencing of P248 (Nuc A, fw), P275 (N-TEV_36AAlinker, fw), P299 (IRES, fw), P292 (QC I of P163 NPT-casette,fw+rv), P293 (QC V of P217 Laccase,fw+rv) and P290 (IgKappa_EreB,fw+rv).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P248
NucA
FR02305834 (O3)
-
P275
N-TEV_36AAlinker
FR02305835 (O3)
P299
IRES
FR02305825 (O3)
-
P292
QC I NPT-casette
FR02305824 (O3)
FR02305823 (O4)
P293
QC V Laccase
FR02305822 (O3)
FR02305821 (O4)
P290
IgKappa_EreB
FR02305820 (O3)
FR02305819 (O4)
Tuesday, June 18th
[Expand] Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275
Investigator: Rosario
Aim of the study: Picking of E. coli XL1 blue transformated with P160, P264, P274 and P275.
Operational sequence:
Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
2 colonies were picked for every transformation product.
[Expand] Transformation of E. coli XL1 blue with F77+F105 (NLS in pSB1C3 + mature thermonuclease) and P299 Retrafo
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1 blue with P217 (QC of 16.05.2013), P163 (QC of 16.05.2013) and P160, P264, P274 and P275 for retransformations.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB)
Investigator: Louise
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with F59+F103 (Strep-TEV-Linker in PsB1C3) and F59+F74 (Serk-Sigp in PsB1C3) and P7 (PhyB).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
P7 (PhyB) clone 1
P300
P7 (PhyB) clone 2
P301
F59+F74 (Serk-Sigp in PsB1C3) clone 1
P302
F59+F74 (Serk-Sigp in PsB1C3) clone 2
P303
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 1
P304
F59+F103 (Strep-TEV-Linker in PsB1C3) clone 2
P305
[Expand] Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI
Investigator: Louise, Rosario, Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of P300-P301 (PhyB), P302-P303 (Serk-Sigp in PsB1C3) and P304-P305 (Strep-TEV-Linker in PsB1C3) with XbaI and AgeI.
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
AgeI
0.25 µl
XbaI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P300-P305)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane:
Digestion of P300 with XbaI/AgeI
Digestion of P301 with XbaI/AgeI
Digestion of P302 with XbaI/AgeI
Digestion of P303 with XbaI/AgeI
Digestion of P304 with XbaI/AgeI
Digestion of P305 with XbaI/AgeI
1 kb DNA Ladder
100 bp DNA Ladder
Result:
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI
Investigator: Louise, Flo, Jeff, Rosario
Aim of the experiment: Preparative digestion and gelelectrophoresis of P212 (SERK-TMD) with NgoMIV/SpeI, P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI, P303 (SERK-SigP) with AgeI/SpeI and P304 (StrepTEV-Linker) with AgeI/SpeI.
Procedure:
Batch for preparative digestion of P212 (SERK-TMD) with NgoMIV/SpeI
volume
reagent
20 µl
P212
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI
volume
reagent
20 µl
P238
4 µl
CutSmart Buffer
1 µl
NgoMIV(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P303 (SERK-SigP) with AgeI/SpeI
volume
reagent
20 µl
P303
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P304 (StrepTEV-Linker) with AgeI/SpeI
volume
reagent
20 µl
P304
4 µl
CutSmart Buffer
1 µl
AgeI(20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and P238, P303 and P304 were loaded on a 1% agarose gel, while P212 was loaded on a 2% agarose gel for preparative gelelectrophoresis.
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane:
Digestion of P238 (SERK-TMD_Linker_GFP) with NgoMIV/SpeI
1 kbp DNA ladder
Digestion of P303 (SERK-SigP) with AgeI/SpeI
Digestion of P304 (StrepTEV-Linker) with AgeI/SpeI
Result:
as expected
as expected
as expected
Lane:
1 kbp DNA ladder
Digestion of P212 (SERK-TMD) with NgoMIV/SpeI
100 bp DNA Ladder
Result:
as expected
The DNA was extracted from the gel with QIAquick Gel Extraction Kit, QIAGEN
Aim of the experiment: Ligation of F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1).
Procedure:
Ligation batch for F109+F86 (SERK-SigP + EreB), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
5.57 µl
F86 (31.0 ng/µl, 1254 bp)
10.51 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F87 (SERK-SigP + NanoLuc), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
6.89 µl
F87 (10.2 ng/µl, 510 bp)
9.19 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F88 (SERK-SigP + SpyCatcher), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
5.37 µl
F88 (8.7 ng/µl, 339 bp)
10.71 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F89 (SERK-SigP + SpyTag), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
12.0 µl
F89 (6.8 ng/µl, 39 bp)
4.08 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F97 (SERK-SigP + XylE), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
6.52 µl
F97 (19.4 ng/µl, 918 bp)
9.56 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F107 (SERK-SigP + SERK-TMD), 100 ng vector used, insert:vector = 3:1
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
5.38 µl
F107 (4.8 ng/µl, 186 bp)
10.70 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), 100 ng vector used, insert:vector = 3:1
volume
reagent
1.38 µl
F110 (72.4 ng/µl, 2143 bp)
5.53 µl
F108 (23.6 ng/µl, 933 bp)
10.09 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] QuikChange mutagenesis to remove forbidden restriction sites - SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)
Investigator: Jeff
Aim of the experiment: Removal of the last existing forbidden restriction site from P217 (Laccase) and the first one from P162 (NPT-Cassette).
Procedure: Quikchange - PCR
Reaction batch for P217 (Laccase)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid template (P217 - 1:100 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O74 (=10 µM)
1 µl
1:10 dilution of O75 (=10 µM)
18.5 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P163 (NPT-Cassette)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid template (P162 - 1:50 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O70 (10 µM)
1 µl
1:10 dilution of O71 (10 µM)
18.5 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P251 (Laccase) and P163 (NPT-Cassette)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
4 min
4 °C
hold
After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.
[Expand] Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMV of P217 (Laccase RFC10) and SDMI of P162 (NPT-Cassette RFC10)
Investigator: Jeff
Aim of the experiment:
Procedure:
4.5 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 40 min at an 1% agarose gel.
Lane:
1 kbp DNA ladder
DpnI digestion of P163QCI
DpnI digestion of P217QCV
Result:
as expected
as expected
[Expand] Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F109+F86 (SERK-SigP + EreB), F109+F87 (SERK-SigP + NanoLuc), F109+F88 (SERK-SigP + SpyCatcher), F109+F89 (SERK-SigP + SpyTag), F109+F97 (SERK-SigP + XylE), F109+F107 (SERK-SigP + SERK-TMD) and F110+F108 (Strep-tag-TEV-site-linker + SERK-TMD_8AAlinker_GFPmut1), with DpnI digested QuikChange products P162QCI and P217QCV, and P212 (ReTraFo), P238 (ReTraFo).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/10 µl of ligation products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Sequencing of P294 (AlcR)
Investigators: Rosario
Aim of the experiment: Sequencing of P294 (AlcR).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P294
AlcR
FR02305817 (O3)
FR02305818(O4)
Wednesday, June 19th
[Expand] QuikChange mutagenesis to remove forbidden restriction sites - SDMI of P294 (AlcR)
Investigator: Florian - Master of QuikChange 2.0
Aim of the experiment: Removal of the first forbidden restriction site from P294 (AlcR).
Procedure: Quikchange - PCR
Reaction batch for P294 (AlcR)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid template (P294 - 1:50 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O80 (=10 µM)
1 µl
1:10 dilution of O81 (=10 µM)
18.5 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P294 (AlcR)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
19
95 °C
30 s
52 °C
1 min
68 °C
5 min
4 °C
hold
After the PCR, the Quickchange products were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.
[Expand] Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)
Investigator: Florian
Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR).
Procedure:
4 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.
Lane:
1 kbp DNA ladder
DpnI digestion of P294QCI
Result:
only Primers
[Expand] Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA)
Investigator: Florian
Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294QCI and P294 (ReTrafo of FluA).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.
[Expand] Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B)
Investigators: Rosario
Aim of the experiment: Sequencing of P308 (ReTrafo of P264, PhyB_36AALinker_C-TEV in pSB1C3), P310 (ReTrafo of P274, N-TEV_36AALinker in pSB1C3), P303 (SERK-SigP in pSB1C3), P304 (Strep-TEV-Linker in pSB1C3), P305 (Strep-TEV-Linker in pSB1C3) and P301 (ReTrafo of P7, Phytotchrome B).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P308
PhyB_36AALinker_C-TEV in pSB1C3
FR02305816 (O3)
FR02305815 (O4)
P310
N-TEV_36AALinker in pSB1C3
FR02305814 (O3)
P303
SERK-SigP in pSB1C3
FR02305813 (O3)
-
P304
Strep-TEV-Linker in pSB1C3
FR02305812 (O3)
-
P305
Strep-TEV-Linker in pSB1C3
FR02305811 (O3)
-
P301
Phytotchrome B
FR02305810 (O3)
FR02305809 (O4)
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3)
Investigator: Rosario
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with ReTrafo of P160 (SV40 NLS), ReTrafo of P264 (PhyB_36AALinker_C-TEV in pSB1C3), ReTrafo of P274 (N-TEV_36AALinker in pSB1C3) and ReTrafo of P275 (N-TEV_36AALinker in pSB1C3).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
P160 I
P306
P160 II
P307
P264 I
P308
P264 II
P309
P274 I
P310
P274 II
P311
P275 I
P312
P275 II
P313
[Expand] Pouring of CamR gel plates
Investigator: Louise, Volker
Aim of the experiment: Pouring of CamR gel plates
Procedure:
Preparation of 5 l of LB-Medium with agar was performed after standard recipe
[Expand] Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)
Investigator: Rosario
Aim of the experiment: Picking of QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)
Procedure:
Retransformations (P212, P238, P299) 1 clone each
Quickchanges (QCI P162, QCV P217) 2 clones each
Ligation F77+F105 4 clones because of the high number of colonies at F77 negative ctrl
All other Ligations (F110+F108, F109+F86, F109+F87, F109+F88, F109+F89, F109+F97, F109+F107) 2 clones each
Thursday, June 20th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD)
Investigator: Rosario
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QC I of P162 (npt-casette in pSB1C3), QC V of P217 (Laccase), ReTrafo of P212, ReTrafo of P238, ReTrafo of P299. F77+F105 (NucA+NLS), F110+F108 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), F109+F86 (SERK-SigP+EreB), F109+F87 (SERK-SigP+NanoLuc), F109+F88 (SERK-SigP+SpyCatcher), F109+F89 (SERK-SigP+SpyTag), F109+F97 (SERK-SigP+xylE), F109+F107 (SERK-SigP+SERK-TMD).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
QC I P162 I
P315
QC I P162 II
P316
QC V P217 I
P317
QC V P217 II
P318
P212 ReTrafo
P319
P238 ReTrafo
P320
P299 ReTrafo
P321
F77+F105 I
P322
F77+F105 II
P323
F77+F105 III
P324
F77+F105 IV
P325
F110+F108 I
P326
F110+F108 II
P327
F109+F86 I
P328
F109+F86 II
P329
F109+F87 I
P330
F109+F87 II
P331
F109+F88 I
P332
F109+F88 II
P333
F109+F89 I
P334
F109+F89 II
P335
F109+F97 I
P336
F109+F97 II
P337
F109+F107 I
P338
F109+F107 II
P339
[Expand] Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222
Investigator: Johanna, Andi
Aim of the experiment: Retransformation of E. coli XL1 blue with P170, P111, P314, P160, P275, P244, P240, P212, P208, P114, P217, P204, P213 and P222.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
2 µl of ddH2O was added to empty tubes
2 µl of DNA was added to 100 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol (P170, P111, P160, P275, P244, P240, P212, P208, P217, P204, P213, P222) and ampicillin plates (P314, P114).
[Expand] Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1)
Investigators: Rosario
Aim of the experiment: Sequencing of P320 (ReTrafo of P238,SERK-TMD_8AAlinker_GFPmut1).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
Label
Name
Barcode1
Barcode2
P308
SERK-TMD_8AAlinker_GFPmut1
FR02305808 (O3)
FR02305807 (O4)
[Expand] Preparation of media for competent cells
Investigator : Johanna, Louise
Aim of the experiment:Preparation of media for competent cells
Procedure:
Picking of one colony of overnight stem culture of non competent XL1-Blue E.Coli in 50 ml LB-Medium without antibiotics (under the sterile bench)
this was incubate overnight at 37 °C
Batch for 1l of a 0.1 M MgCl2-solution
amount
reagent
20.33 g
MgCl2 (M=203.3 g/mol)
1 l
ELGA H2O
Batch for 500ml of a 50 mM CaCl2-solution
amount
reagent
3.67 g
CaCl2 (M=147.02 g/mol)
500 ml
ELGA H2O
Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
amount
reagent
0.368 g
CaCl2 (M=147.02 g/mol)
0.458 g
Glycerin (density=1.26)
50 ml
ELGA H2O
Autoclave each bottle of solution and leave each in a 4°C room.
[Expand] QuikChanges QC pActin and QC1 AlcR
Investigator : Andi
Aim of the experiment: QuikChanges QC pActin (P114) and QC1 AlcR (P294)
Procedure: Quickchange - PCR
Reaction batch 1 for P114 (pActin) with Pfu Ultra II
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P114 undiluted)
4.5 ng in 25 µl batch
1 µl
1:10 dilution of O76 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
1 µl
1:10 dilution of O77 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
18 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P114 (pActin)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
3
52 °C
1 min
4
68 °C
6.5 min
5
4 °C
hold
Reaction batch 2 for P114 (pActin) with Q5 Polymerase Mastermix
volume
reagent
Additional information
12.5 µl
2x QS Polymerase Mastermix
1 µl
Plasmid template (P114 - 1:10 dilution)
0.45 ng in 25 µl batch
1.25 µl
1:10 dilution of O76 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
1.25 µl
1:10 dilution of O77 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
9 µl
ddH2O
PCR cycling parameters - P114 (pActin) with Q5 Polymerase
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
3
55 °C
1 min
4
72 °C
3.25 min
5
4 °C
hold
Reaction batch for P294 (AlcR) with Pfu Ultra II
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
0.5 µl
Plasmid template (P294 dilutsion 1:100)
2.79 ng in 25 µl batch
1 µl
1:10 dilution of O76 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
1 µl
1:10 dilution of O77 (=10 µM)
0.5 µl Primer + 4.5 µl ddH2O
18.5 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P294 (AlcR)
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
3
52 °C
1 min
4
68 °C
6.5 min
5
4 °C
hold
After the PCR, the Quickchange products of P114 (pActin) with Q5 Mastermix and P294 (AlcR)were mixed with 1 µl of DpnI (10 U/µl) and incubated at 37 °C for 1 h.
After the PCR, the Quickchange products of P114 (pActin) with PfU II Polymerase was stored in the Cycler at 4 °C over night
[Expand] Transformation of E. coli XL1-blue with DpnI digested QuikChange product P114 (pActin) and P294 (AlcR)
Investigator: Master of Quickchange, Gel-Penetrator
Aim of the experiment: Transformation of E. coli XL1-blue with DpnI digested QuikChange product P294 (AlcR) QCI and P114 (pActin).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/2 µl of plasmid DNA was added to 200 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.
[Expand] PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase
Investigator: Andi, Johanna
Aim of the experiment: PCR of P317 (Laccase) with Q5 Mastermix and Herculase Polymerase
Procedure:
Operational sequence:
Reaction batch with Herculase Polymerase
volume
reagent
10 µl
5x Herculase Fusion Polymerase reaction buffer
2.5 µl
10 mM dNTPs
1.25 µl
10 µM Forward Primer O24 (Bpul_for)
1.25 µl
10 µM Reverse Primer O25 (Bpul_rev)
1 µL
Herculase II Fusion Polymerase
1.5 µl
Plasmid DNA (P317)- 1:500 dilution
0.5 µl
DMSO
32 µL
ddH2O Water
=50 µL
TOTAL
Reaction batch with Q5 Mastermix
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O24 (Bpul_for)
2.5 µl
10 µM Reverse Primer O25 (Bpul_rev)
1 µl
Plasmid DNA (P317)- 1:500 dilution
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme:
Initial denaturation
94 °C
30 s
30 cycles
94 °C
30 s
51 °C
60 s
68 °C
90 s
Final extension
68 °C
5 min
Hold
4 °C
infinite
After the PCR has finished, the product was hold at 4 °C in the Cycler
[Expand] Analytical gelelectrophoresis of the DpnI digested products of the performed QuickChange of SDMI of P294 (AlcR)and P114 (pActin)
Investigator: Andi, Johanna
Aim of the experiment: Analytical gelelectrophoresis of the DpnI digested products of the performed QuikChange of SDMI of P294 (AlcR)and P114 (pActin.)
Procedure:
9 µl of each of the DpnI digested QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.
Lane:
100 bp DNA ladder
DpnI digestion of P294QCI
DpnI digestion of P114QCI
100 bp DNA ladder
Result:
expected result
only Primers
[Expand] Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)
Investigator: Rosario, Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P315 and P316 (QC I P162 NPT-casette), P162 (npt-casette in pSB1C3), P317 and P318 (QC V P217 Laccase), P217 (Laccase), P322 (F77+F105 NucA+NLS), P248 (Thermonuc in pSB1C3), P320 (SERK-TMD_8AAlinker_GFPmut1), P326+P327 (Strep-tag-TEV-site-linker+SERK-TMD_8AAlinker_GFPmut1), P328+P329 (SERK-SigP+EreB), P157 (EreB), P330+P331 (SERK-SigP+NanoLuc), P208 (NanoLuc), P332+P333 (SERK-SigP+SpyCatcher), P213 (SpyCatcher), P334+335 (SERK-SigP+SpyTag), P170 (SpyTag), P336+P337 (SERK-SigP+xylE), P197 (xylE), P338+P339 (SERK-SigP+SERK-TMD), P212(SERK-TMD)
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
PstI
15 µl
ddH2O
2.5 µl
Plasmid DNA
=20 µl
TOTAL
Incubation for 90 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
1 kb DNA Ladder
P315
P316
P162 (Control)
P317
P318
P217 (Control)
P322
P323
P324
P325
P248 (Control)
as expected
as expected
as expected
as expected
as expected
as expected
corrupt
corrupt
corrupt
corrupt
as expected
1 kb DNA Ladder
P334
P335
P170 (Control)
P336
P337
P197 (Control)
P338
P339
P212 (Control)
P320
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
is repeated on other gel
1 kb DNA Ladder
P320 (Control)
P326
P327
as expected
as expected
as expected
1 kb DNA Ladder
P328
P329
P157 (Control)
P330
P331
P208 (Control)
P332
P333
P213 (Control)
P326
P327
as expected
as expected
as expected
as expected
as expected
corrupt
as expected
as expected
corrupt
is repeated on other gel
is repeated on other gel
Friday, June 21st
[Expand] Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)
Investigator: Master of QuikChange
Aim of the experiment: Analytical gelelectrophoresis of the PCR product of P317 (Laccse) with PfU II Polymerase, Herculase Polymerase and the DpnI digested products of the performed QuickChange of P114 (pActin)
Procedure:
9 µl of each of the PCR products were mixed with 1 µl of DNA loading buffer (10x).
9 µl of each of the DpnI digested QuikChange product was mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 50 min at an 1% agarose gel.
Lane:
1000 bp DNA ladder
PCR product of P317 with Q5 Master Mix
PCR product of P317 with Herculase Polymerase
DpnI digestion of P114
Result:
expected result
expected result
expected result
[Expand] Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)
Investigator: Johanna, Louise
Aim of the experiment: Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)
Procedure:
Add Tetracyclin (ratio: 1:1000) to the 2YT-medium
Measure the OD550 of it until OD550=0.5
Put the culture in Falcon tubes (leaving on ice)
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 40 ml 0,1 M MgCl2 solution after removing the supernatants
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 20 ml 50mM CaCl2 solution after removing the supernatants
Incubate the falcon tubes on ice for 30 min
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 2 ml 50mM CaCl2, 15 % v/v Glycerin solution after removing the supernatants. From this step everything needs to be carry out in a 4°C room.
Mix the solutions of each falcon tube and transfer the content in eppis (each with 100 and accordingly 150 µl)
Aim of the experiment: Ligation of F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3)
Procedure:
Ligation batch for F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB)
volume
reagent
3.52 µl
F117 (28.4 ng/µl, 3413 bp)
4.81 µl
F116 (18.2 ng/µl, 996 bp)
8.67 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc)
volume
reagent
1.89 µl
F118 (52.8 ng/µl, 2663 bp)
6.16 µl
F116 (18.2 ng/µl, 996 bp)
8.95nbsp;µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher)
volume
reagent
2.57 µl
F119 (38.0 ng/µl, 2492 bp)
6.59 µl
F116 (18.2 ng/µl, 996 bp)
7.84nbsp;µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag)
volume
reagent
0.8 µl
F120 (62.5 ng/µl, 2192 bp)
3.75 µl
F116 (18.2 ng/µl, 996 bp)
3.96nbsp;µl
ddH2O
1 µl
T4 ligase buffer (10x)
0.5 µl
T4 ligase (1 U/µl)
=10 µl
TOTAL
Ligation batch for F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE)
volume
reagent
0.73 µl
F121 (68.3 ng/µl, 3071 bp)
2.56 µl
F116 (18.2 ng/µl, 996 bp)
5.95nbsp;µl
ddH2O
1 µl
T4 ligase buffer (10x)
0.5 µl
T4 ligase (1 U/µl)
=10 µl
TOTAL
Ligation batch for F123+F124 (Laccase+pSB1C3)
volume
reagent
2.03 µl
F123 (49.3 ng/µl, 2086 bp)
3.80 µl
F124 (58.9 ng/µl, 1555 bp)
11.17nbsp;µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3) and negative controls
Investigator: Jeff, Katrin
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F116+F117 (TEV-Linker-TM-GFP+SERKSigP_EreB), F116+F118 (TEV-Linker-TM-GFP+SERK-SigP_NLuc), F116+F119 (TEV-Linker-TM-GFP+SERK-SigP_SpyCatcher), F116+F120 (TEV-Linker-TM-GFP+SERK-SigP_SpyTag), F116+F121 (TEV-Linker-TM-GFP+SERK-SigP_XylE), F123+F124 (Laccase+pSB1C3), QCI(Insertion) of pActin and negative controls
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol/ampicillin plate.
[Expand] Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw)
Investigators: Louise, Katrin
Aim of the experiment: Sequencing of P303 (SERK-SigP in PsB1C3, fw), P310 (N-TEV_36AALinker in pSB1C3, rev), P248 (Thermonuc in pSB1C3, fw), P312 (N-TEV_36AALinker in pSB1C3, fw), P316 (npt-casette after QCI, fw,rev), P318 (Laccase after QCV, still RFC10, fw, rev), P320 (SERK-TMD_8AAlinker_GFPmut1, fw, rev), P326 (Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1, fw, rev), P328 (SERK-SigP_EreB, fw, rev), P330 (SERK-SigP_NanoLuc, fw), P332 (SERK-SigP_SpyCatcher, fw), P334 (SERK-SigP_SpyTag, fw), P336 (SERK-SigP_XylE, fw, rev), P338 (SERK-SigP_SERK-TMD, fw).
Procedure: Peparation of DNA for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng/µl) and 2 µl sequencing primer 10 µM).
The different genes received the following barcodes:
[Expand] PCR of P61 to get the IRES of poliovirus 1 Mahoney strain
Investigator: Jeff
Aim of the experiment: PCR of P61 to get the IRES of poliovirus 1 Mahoney strain.
Procedure:
Operational sequence:
Reaction batch with Q5 Mastermix:
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O68 (IRES_polio_fw)
2.5 µl
10 µM Reverse Primer O69 (IRES_polio_rv)
1 µl
Plasmid DNA (P61)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
63 °C
30 s
72 °C
20 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
[Expand] Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR
Investigator: Katrin
Aim of the experiment: Picking of Re-Transformations P111 (TEV in pSB1C3, Re-Trafo), P160 (Sv40 NLS in pSB1C3), P114 (pActin), P314 (FluA), Glutathione S-transferase (GST, BBa_K620000), QCI AlcR.
Procedure:
Retransformations 1 clone each
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol (P111, P160) or ampicillin (P114, P314)
Saturday, June 22nd
[Expand] QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII)
Investigator: Jeff
Aim of the experiment: QuikChange of npt-casette (P316, QCII) and of AlcR (P342, QCII).
Procedure:
Reaction batch for P316 (npt-casette after QCI)
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P316 - 1:100 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O72 (=10 µM)
1 µl
1:10 dilution of O73 (=10 µM)
19 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch for P342 (AlcR after QCI):
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P342 - 1:100 dilution)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O82 (10 µM)
1 µl
1:10 dilution of O83 (10 µM)
19 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters - P316 (npt-casette after QCI) and P342 (AlcR after QCI):
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
52 °C
1 min
68 °C
5 min
3
4 °C
hold
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000)
Investigator: Katrin
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P111(Retrafo), QCI AlcR, P114(Retrafo), P314 (Retrafo), P160 (Retrafo), Glutathione S-transferase (GST, BBa_K620000).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
[Expand] Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI, gelelectrophoresis of PCR of IRES
Investigator: Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of QCII AlcR (P341, P342) and control P294 with EcoRI and PstI
Procedure:
Batch
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
PstI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P341, P342; P249)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane:
1 kb DNA Ladder
PCR of IRES
Digestion of P249 with EcoRI and PstI (control)
Digestion of P341 with EcoRI and PstI
Digestion of P342 with EcoRI and PstI
Result:
as expected
as expected
as expected
as expected
[Expand] Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES
Investigator: Katrin
Aim of the experiment: Preparative Digestion of P310 (N-Tev-linker), P249 (Thermonuclease), P160 (SV40 NLS), PCR of IRES
Procedure:
Preparative digestion of P310 (N-Tev-linker)
volume
reagent
20 µl
P310
4 µl
CutSmart Buffer (10x)
1 µl
AgeI (20 U/µl)
1 µl
SpeI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Preparative digestion of P160 (SV40 NLS)
volume
reagent
20 µl
P160
4 µl
CutSmart Buffer (10x)
1 µl
AgeI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Preparative digestion of P249 (Thermonuclease)
volume
reagent
20 µl
P249
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Preparative digestion of PCR of IRES)
volume
reagent
25 µl
PCR product
5 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
SpeI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Name:
P249 dig. NgoMIV + PstI
1kb DNA ladder
P160 dig. AgeI + PstI
P111 dig. SpeI + AgeI
cut out:
Insert(lower band)
Backbone
Backbone
Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit
[Expand] Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3)
Investigator: Katrin
Aim of the experiment: Ligation of F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IREs+pSB1C3)
Procedure:
Ligation batch for F126+F127 (NLS in pSB1C3+Thermonuclease
volume
reagent
7.2 µl
F126 (13.9 ng/µl, 2143 bp)
4.1 µl
F127 (15.2 ng/µl, 447 bp)
5.7 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F125+F96 (N-TEV-linker in pSB1C3+PIF3)
volume
reagent
6.9 µl
F125 (14.5 ng/µl, 2554 bp)
2.3 µl
F96 (15.1 ng/µl, 297 bp)
7.8nbsp;µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F124+F41 (IREs+pSB1C3)
volume
reagent
1.3 µl
F124 (70.8 ng/µl, 628 bp)
1.2 µl
F41 (81.1 ng/µl, 2086 bp)
14.5nbsp;µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls
Investigator: Volker
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F126+F127 (NLS in pSB1C3+Thermonuclease), F125+F96 (N-TEV-linker in pSB1C3+PIF3), F125+F98 (N-TEV-linker in pSB1C3+PIF6), F124+F41 (IRES+pSB1C3) and negative controls
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)
Investigator: Katrin
Aim of the experiment: Picking of F116+F121, F116+F117, F123+F124, F116+F119, F116+F120, F116+F118, QCI (insertion of P114 (pActin)
Procedure:
2 clones were picked from each plate
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol or ampicillin (QCI (insertion of P114 (pActin))
Sunday, June 23rd
[Expand] QuikChange II of pActin (P359, P360)
Investigator: Katrin
Aim of the experiment: QuikChangeII of pActin (P359, P360)
Procedure:
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P359, P360)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O76 (=10 µM)
1 µl
1:10 dilution of O77 (=10 µM)
19 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
52 °C
1 min
68 °C
6 min
3
4 °C
hold
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)
Investigator: Katrin
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue F123+F124, F116+F117, F116+F119, F116+F118, F116+F121, F116+F120, QCI pActin (P114)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
[Expand] Analytical digestion and gelelectrophoresis of P347-P358
Investigator: Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P347-P358
Procedure:
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
PstI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P347-P358)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane:
1 kb DNA Ladder
Digestion of P347 with EcoRI and PstI
Digestion of P348 with EcoRI and PstI
Digestion of P349 with EcoRI and PstI
Digestion of P350 with EcoRI and PstI
Digestion of P351 with EcoRI and PstI
Digestion of P352 with EcoRI and PstI
Digestion of P353 with EcoRI and PstI
Digestion of P354 with EcoRI and PstI
Result:
Lane:
1 kb DNA Ladder
Digestion of P355 with EcoRI and PstI
Digestion of P356 with EcoRI and PstI
Digestion of P357 with EcoRI and PstI
Digestion of P358 with EcoRI and PstI
Result:
[Expand] Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette
Investigator: Katrin
Aim of the experiment: Dpn1 Digestion and transformation of E. coli XL1-blue with QCII of AlcR and QCII of npt-cassette
Procedure:
1 µl of Dpn1 was added to the reaction, the batch was incubated for one hour at 37°C
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Picking of F125+F98, F125+F96, F128+F41
Investigator: Katrin
Aim of the experiment:Picking of F125+F98, F125+F96, F128+F41
Procedure:
2 clones were picked from each plate
Resuspendsion in 4 ml LB-medium with 4 µl chloramphenicol
F126+F127 was put back into the incubator because there were no colonies visible
[Expand] Sequencing of P35, P143, P359, P360, P249
Investigators: Katrin
Aim of the experiment: Sequencing of P35, P143, P359, P360, P249
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
P35
FR02305786 (O4)
P143
FR02305785 (O92)
FR02305784 (O93)
P359
FR02305783 (O62)
FR02305782 (O63)
P360
FR02305781 (O62)
FR02305780 (O63)
P249
FR02305779 (O3)
FR02305778 (O4)
Week 10
Monday, June 24th
[Expand] Miniprep of ligations F125+F98 (N-TEV-linker in pSB1Cs + PIF6), F125+F96 (N-TEV-linker in pSB1Cs + PIF3) and F128+F41 (PCR of IRES + pSB1C3 backbone) + analytical gel
Investigator: Johanna
Aim of the experiment: Preparation of ligation products F125+F98, F125+F96 and F128+F41
Procedure:
Miniprep according to QIAGEN QIAprep Kit
label
content
conc. in ng/µl
P361
N-TEV-linker (in pSB1C3) + PIF6
163.2
P362
N-TEV-linker in (in pSB1C3) + PIF6
183.5
P363
N-TEV-linker in (in pSB1C3) + PIF3
159.6
P364
N-TEV-linker in (in pSB1C3) + PIF3
200.1
P365
PCR of IRES + (in pSB1C3) backbone
136.0
P366
PCR of IRES + (in pSB1C3) backbone
153.7
Analytical digestion with EcoRI and PstI
reagent
volume
CutSmart buffer
2 µl
EcoRI
0.25 µl
PstI
0.25 µl
ddH2O
15 µl
plasmid DNA
2.5 µl
Total
20 µl
60 min incubation, 37 °C
Gel elektrophoresis on 1% agarose gel
1 kbp DNA ladder
P361
P362
P363
P364
P365
P366
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Dpn1 digestion and transformation of QCII of pActin (insertion) + analytical Gel
Investigator: Jeff
Aim of the experiment: Dpn1 digestion and transformation of QCII of pActin (insertion)
Procedure:
1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new ampicillin plate.
0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid
Electrophoresis on 1% agarose gel
1 kb dna ladder
P359
P360
successful
successful
[Expand] Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)
Investigator: Florian
Aim of the experiment: Sequencing of P266 (IgKappa-SigP_Nanoluc), P268 (IgKappa-SigP_XylE), P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P361 (N-TEV-linker+PIF6), P364 (N-TEV-36AAlinker-PIF3) and P366 (IRES)
Procedure: DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The samples received the following barcodes:
Label
Barcode1
Barcode2
P266
FR02305766 fw
FR02305767 rv
P268
FR02305768 fw
FR02305769 rv
P270
FR02305770 fw
P272
FR02305771 fw
P361
FR02305772 fw
FR02305773 rv
P364
FR02305774 fw
FR02305775 rv
P366
FR02305776 fw
FR02305777 rv
[Expand] Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)
Investigator: Johanna
Aim of the experiment: Retransformation of P270 (IgKappa-SigP_SpyCatcher in pSB1C3), P272 (IgKappa-SigP_SpyTag in pSB1C3), P308 (PhyB_36AALinker_C-TEV in pSB1C3) and P366 (IRES in pSB1C3)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1.5 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of cell suspension were plated on chlorampenicol agar
The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.
Aim of the experiment: Ligation of F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES).
Procedure:
Ligation batch for F92 + F130 (IgKappa-SigP + Laccase)
volume
reagent
0.86 µl
F92 (116.7 ng/µl, 2144 bp)
7.55 µl
F130 (28.3 ng/µl, 1527 bp)
8.59 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109 + F130 (SERK-SigP + Laccase)
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2177 bp)
7.44 µl
F130 (28.3 ng/µl, 1527 bp)
8.64 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES)
volume
reagent
0.82 µl
F113 (122.2 ng/µl, 5281 bp)
2.17 µl
F131 (16.4 ng/µl, 627 bp)
14.01 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F115 + F131 (TEV + IRES)
volume
reagent
1.76 µl
F115 (56.9 ng/µl, 2794 bp)
4.1 µl
F131 (16.4 ng/µl, 627 bp)
11.14 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD)
Investigator: Florian, Jeff
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and negative controls and BioBrick BBa_K325909 (lucABCDEG under pBAD).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of QuikChange products/2 µl of plasmid DNA was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES)
Investigator: Flo, Jeff
Aim of the experiment: Preparative digestion of P308 (PhyB_36AAlinker_C-TEV), P348 (Laccase RFC25) and P366 (polioviral IRES).
Procedure:
volume
reagent
14 µl
P308
4 µl
CutSmart Buffer (10x)
1 µl
SpeI-HF (20 U/µl)
1 µl
PstI-HF (20 U/µl)
20 µl
ddH2O
=40 µl
TOTAL
volume
reagent
14 µl
P348
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV (20 U/µl)
1 µl
SpeI-HF (20 U/µl)
20 µl
ddH2O
=40 µl
TOTAL
volume
reagent
20 µl
P366
4 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated for 2.5 h at 37 °C.
4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.
Preparative geleletrophoresis was performed at 90 V for 60 min.
Lane
Digestion of P308 with SpeI & PstI
1 kbp DNA ladder
Digestion of P348 with NgoMIV & SpeI
Digestion of P366 with XbaI & PstI
result
2 further unexpected bands; seems that PstI is contaminated with a prefix restriction enzyme; used PstI enzyme batch is discareded; upper band was cut out
as expected; lower band was cut out
as expected; lower band was cut out
[Expand] Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII
Investigator: Jeff
Aim of the experiment: Picking of F126+F127 (NLS+ThermoNuc), AlcR QCII and npt-cassette QCII for plasmid preparation
Procedure:
F126+F127 (NLS+ThermoNuc) 7 clones due to high number of colonies on negative ctrl
AlcR QCII and npt-cassette QCII 2 clones each
Tuesday, June 25th
[Expand] Sequencing of P344 pASK-Flua(Trippelmutante)
Investigator: Jeff
Aim of the experiment: Sequencing of P344. Concentration was determined by Nanodop to 132 ng/µl
Procedure:
DNA samples were prepared according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The samples received the following barcodes:
Label
Barcode1
P344 with F83 (primer from Skerra lab! not fragment 83 from iGEM!)
FR02305765
[Expand] Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)
Investigator: Jeff, Rosario
Aim of the experiment: Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
empty tube was washed with 2 µl water and the volume was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
100 µl of cell suspension were plated on chlorampenicol agar
The rest of the cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium; the concentrated cell suspension was plated on another chlorampenicol agar plate.
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease)
Investigator: Louise
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QCII of P316 (npt-casette in pSB1C3), QCII of P342 (AlcR in pSB1C3) and F126+F127 (NLS in pSB1C3 + Thermonuclease).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
F126+F127 clone 1
P367
F126+F127 clone 2
P368
F126+F127 clone 3
P369
F126+F127 clone 4
P370
F126+F127 clone 5
P371
F126+F127 clone 6
P372
F126+F127 clone 7
P373
QCII npt clone 1
P374
QCII npt clone 2
P375
QCII AlcR clone 1
P376
QCII AlcR clone 2
P377
[Expand] Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI
Investigator: Louise, Rosario, Florian
Aim of the experiment:Analytical digestion and gelelectrophoresis of P367-P373 (NLS in pSB1C3 + Thermonuclease) with EcoRI and PstI, P374-P375 (QCII of npt casette) with XbaI and PstI, P376-P377 (QCII of AlcR) with XbaI and SpeI.
Procedure:
Master-Mix for P367-P373 and control P249
volume
reagent
20 µl
CutSmart Buffer (10x)
2.5 µl
EcoRI
2.5 µl
PstI
150 µl
ddH2O
=175 µl
TOTAL
Master-Mix for P374-P375 and control P316
volume
reagent
8 µl
CutSmart Buffer (10x)
1 µl
XbaI
1 µl
PstI
60 µl
ddH2O
=70 µl
TOTAL
Master-Mix for P376-P377 and control P342
volume
reagent
8 µl
CutSmart Buffer (10x)
1 µl
XbaI
1 µl
SpeI
60 µl
ddH2O
=70 µl
TOTAL
Batch for analytical digestion of P367-P377 and controls
volume
reagent
17.5 µl
Master-Mix
2.5 µl
Plasmid DNA
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane
1 kbp DNA ladder
Digestion of P367 with EcoRI and PstI
Digestion of P368 with EcoRI and PstI
Digestion of P369 with EcoRI and PstI
Digestion of P370 with EcoRI and PstI
Digestion of P371 with EcoRI and PstI
Digestion of P372 with EcoRI and PstI
Digestion of P373 with EcoRI and PstI
Digestion of P249 with EcoRI and PstI
result
as expected
as expected
not as expected
not as expected
as expected
as expected
not as expected
as expected
Lane
1 kbp DNA ladder
Digestion of P316 with XbaI and PstI
Digestion of P374 with XbaI and PstI/SpeI
Digestion of P374 with XbaI and PstI/SpeI
Digestion of P375 with XbaI and PstI/SpeI
Digestion of P375 with XbaI and PstI/SpeI
Digestion of P376 with XbaI and SpeI
Digestion of P377 with XbaI and PstI/SpeI
Digestion of P377 with XbaI and PstI/SpeI
Digestion of P342 with XbaI and SpeI
result
not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
not as expected, maybe digestion with EcoRI instead of XbaI, has to be repeated
as expected
not fully digested
not fully digested
as expected
[Expand] Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI
Investigator: Louise, Florian
Aim of the experiment: Preparative digestion and gelelectrophoresis of P26 (Dnb1c Promotor) and P35 (RD29 Promotor) with EcoRI and KpnI .
Procedure:
Batch for P26
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer
1 µl
EcoRI
1 µl
KpnI
14 µl
ddH2O
=40 µl
TOTAL
Batch for P35
volume
reagent
7 µl
Plasmid DNA
4 µl
CutSmart Buffer
1 µl
EcoRI
1 µl
KpnI
27 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and the samples were loaded on a 1% agarose gel.
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane
Digestion of P26 with EcoRI and KpnI
100 bp DNA ladder
Digestion of P26 with EcoRI and KpnI
1 kbp DNA ladder
result
not as expected, no band was cut out
as expected; upper band was cut out, F132
[Expand] Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES)
Investigator: Rosario
Aim of the experiment: Picking of ligation products F92 + F130 (IgKappa-SigP + Laccase), F109 + F130 (SERK-SigP + Laccase), F113 + F131 (PhyB_36AA-Linker_C-TEV + IRES), F115 + F131 (TEV + IRES) and BioBrick BBa_K325909 (lucABCDEG under pBAD) and Retransformation of P270 (IgKappa-SigP_SpyCatcher), P272 (IgKappa-SigP_SpyTag), P308 (PhyB_36AALinker_C-TEV) and P366 (IRES).
Procedure:
2 clones were picked for each ligation product and for the biobrick
1 clone was picked for each ReTrafo
Wednesday, June 26th
[Expand] Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366, lucBB
Investigator: Ingmar
Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with P270, F113+F131, F109+F130, P308, F115+F131, F92+F130, F115+F131, P272, P366 and lucBB.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
The resulting DNA were eluted in tubes labeled as follows:
Content
New Tube
P270 Retrafo
P379
F113+F131
P380
F109+F130
P381
P308 Retrafo
P382
F115+F131
P383
F92+F130
P384
F113+F131
P385
F115+F131
P386
F92+F130
P387
P272 Retrafo
P388
P366
P389
luc BB
P390
F109+F130
P391
luc BB
P392
[Expand] Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P374-P375 (QC II of npt-casette) and P316 (QC I of npt-casette) as a reference with XbaI and PstI
Procedure:
volume
reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
XbaI
0.25 µl
PstI
15 µl
ddH2O
2.5 µl
Plasmid DNA (P316,P374,P375)
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane:
1000 bp DNA ladder
Digestion of P316 with XbaI and PstI
Digestion of P374 with XbaI and PstI
Digestion of P375 with XbaI and PstI
Result:
corrupt
corrupt
corrupt
[Expand] Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3)
Investigator: Rosario
Aim of the experiment: Picking of Retransformation of P35 (Viability sensor with RFP) and P249 (Thermonuc in pSB1C3).
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Aim of the experiment: Sequencing of P387 (IgkKappa-SigP_Laccase (QCV, RFC25)), P391 (SERK-SigP_Laccase (QCV, RFC25))and P355 (SERK-SigP_XylE_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 clone 2).
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
P387
FR02305758 (O3)
FR02305759 (O4)
P391
FR02305760 (O3)
P355
FR02305761 (O3)
FR02305762 (O4)
[Expand] Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)
Investigator: Katrin
Aim of the experiment: Re-Transformation of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
150 µl of competent cells were added to the tubes containing about 1 µl of plasmid DNA and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.
[Expand] PCR of P143 (pLuc-Actin, Amplification of pActin)
Investigator: Rosario, Flo
Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin).
Procedure:
Operational sequence:
Reaction batch with Q5 Mastermix:
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl
10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl
Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
62 °C
30 s
72 °C
45 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
[Expand] Analytical gelelectrophoresis of F133
Investigator: Jeff
Procedure: Analytical gelelectrophoresis of F133 (PCR product of F143 with O96 & O97) to check the quality of the performed PCR.
Procedure:
4.5 µl of F133 were mixed together 0.5 µl of DNA loading buffer (10x)
Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.
Lane:
1000 bp DNA ladder
F133
Result:
as expected; but byproduct visible
[Expand] Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P381, P391, P347, P384, P387, P380, P385, P309, P383, P386, P390, P392 with EcoRI and PstI
Volume
Reagent
2 µl
CutSmart Buffer (10x)
0.25 µl
EcoRI
0.25 µl
PstI
15 µl
ddH2O
2.5 µl
Plasmid DNA
=20 µl
TOTAL
Incubation for 60 min at 37 °C.
Analytical gelelectrophoresis was performed at 90 V for 60 min
Lane:
1000 bp DNA ladder
Digestion of P381 with EcoRI and PstI
Digestion of P391 with EcoRI and PstI
Digestion of P347 with EcoRI and PstI
Digestion of P384 with EcoRI and PstI
Digestion of P387 with EcoRI and PstI
Result:
as expected
as expected
not fully digested
as expected
as expected
Lane:
1000 bp DNA ladder
Digestion of P380 with EcoRI and PstI
Digestion of P385 with EcoRI and PstI
Digestion of P309 with EcoRI and PstI
Digestion of P383 with EcoRI and PstI
Digestion of P386 with EcoRI and PstI
Digestion of P390 with EcoRI and PstI
Digestion of P392 with EcoRI and PstI
Result:
as expected
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S)
Investigator: Flo, Rosario
Aim of the experiment: Preparative digestion and gelelectrophoresis of P391 (SERK-SigP_Laccase), P326 (Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1), P385 (PhyB_36AAlinker_C-TEV_IRES), P361 (N-TEV_36AAlinker_PIF6), P364 (N-TEV_36AAlinker_PIF3), P266 (IgKappa-SigP_NanoLuc), P388 (IgKappa-SigP_SpyTag), P247 (SpyCatcher), P379 (IgKappa-SigP_SpyCatcher), P374 (npt-casette) and P165 (t35S).
Procedure:
Batch for preparative digestion of effectors (P328, P330, P332, P334, P336, P338) with AgeI and PstI)
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer (10x)
1 µl
Enzyme 1 (20 U/µl)
1 µl
Enzyme 2 (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Plasmid
Name
Enzyme 1
Enzyme 2
P391
SERK-SigP_Laccase
AgeI
PstI
P326
Strep-tag-TEV-linker_SERK-TMD_8AAlinker_GFPmut1
NgoMIV
PstI
P385
PhyB_36AAlinker_C-TEV_IRES
SpeI
PstI
P361
N-TEV_36AAlinker_PIF6
XbaI
PstI
P364
N-TEV_36AAlinker_PIF3
XbaI
PstI
P266
IgKappa-SigP_NanoLuc
EcoRI
AgeI
P388
IgKappa-SigP_SpyTag
AgeI
PstI
P247
SpyCatcher
EcoRI
NgoMIV
P379
IgKappa-SigP_SpyCatcher
AgeI
PstI
P374
npt-casette
EcoRI
XbaI
P165
t35S
EcoRI
SpeI
Incubation for 2.5 h at 37 °C.
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane
Digestion of P391 with AgeI & PstI
1kb DNA ladder
Digestion of P326 with NgoMIV & PstI
Digestion of P385 with SpeI & PstI
Results
sideproducts?!; upper band was cut out
as expected, lower band was cut out
as expected
Lane
Digestion of P361 with XbaI & PstI
1kb DNA ladder
Digestion of P364 with XbaI & PstI
Digestion of P266 with EcoRI & AgeI
Results
as expected, lower band was cut out
as expected, lower band was cut out
as expected, lower band was cut out
Lane
Digestion of P388 with AgeI & PstI
1kb DNA ladder
Digestion of P382 with NgoMIV & PstI
Digestion of P247 with EcoRI & NgoMIV
Results
as expected
not expected, wrong plasmid
as expected
Lane
Digestion of P379 with AgeI & PstI
1kb DNA ladder
Digestion of P374 with EcoRI & XbaI
Digestion of P165 with EcoRI & SpeI
Results
as expected
as expected
as expected, but only little DNA
Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit
Negative control was prepared by replacing the volume of the insert by ddH2O.
The ligation was performed overnight at 16 °C.
Friday, June 28th
[Expand] Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1-blue with P338(Ig-Kappa-SigP_SpyTag, Retrafo), F135+F134(SERK-SigP_Laccase+Strep-TEV_linker_SERK-TMD_linker_GFP), F136+F138(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF3), F136+F137(PhyB_linker_C-TEV_IRES+N-TEV_linker_PIF6), F143+F144(npt-Cassette+Terminator) and negative controls (F135, F136, F143)
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed, for re-transformation, 1 µl of P388 was added to 150 µl of competent cells
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] QuikChange III of AlcR (P376)
Investigator: Andi, Johanna
Aim of the experiment: QuikChange III of AlcR (P376)
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P376), (1:120 diluted)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O84 (=10 µM)
1 µl
1:10 dilution of O85 (=10 µM)
19 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
52 °C
1 min
68 °C
5 min
3
4 °C
hold
[Expand] Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)
Investigator: Katrin
Aim of the experiment: Preparative digestion and gelelectrophoresis of P388 (Ig-Kappa-SigP_SpyTag), P246 (Nanoluc), F133 (PCR of pActin)
Procedure:
reaction batch for P388(Ig-Kappa-SigP_SpyTag)
volume
reagent
20 µl
P388
4 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
reaction batch for P246(Nanoluc)
volume
reagent
20 µl
P246
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV-HF (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
reaction batch for F133(PCR of pActin)
volume
reagent
25 µl
F133
5 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Reaction batches were incubated for 3 h at 37 °C.
4 µl DNA loading buffer (10x) were added to the reaction batches after the incubation time and were loaded on a 1% agaorose gel.
Preparative geleletrophoresis was performed at 90 V for 60 min.
Lane
Digestion of F133 with XbaI & PstI
1 kbp DNA ladder
Digestion of P246 with NgoMIV,PstI
Digestion of P388 with AgeI, PstI
result
as expected (but smaller fragment visible), upper band was cut out
as expected, lower band was cut out
as expected
[Expand] Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)
Investigator: Katrin
Aim of the experiment: Ligation of F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3)
Procedure:
Ligation batch for F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc)
volume
reagent
0.90 µl
F146 (111.2 ng/µl, 2189 bp)
3.99 µl
F147 (17.5 ng/µl, 510 bp)
12.11 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F145+F42 (pActin + pSB1C3) (batch: 30 ng vector!)
volume
reagent
23.35 µl
F145 (2.5 ng/µl, 1353 bp)
0.41 µl
F42 (73.5 ng/µl, 2086 bp)
2.7 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=27 µl
TOTAL
Negative controls were prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature.
[Expand] Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)
Investigator: Katrin
Aim of the experiment: Picking of ReTrafos of E. coli XL1-blue with P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc
Procedure:
one clone per plate was picked
4 ml LB-medium, 4 µl chloramphenicol
[Expand] Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel
Investigator: Katrin
Aim of the experiment: Dpn1 digestion and transformation of QCIII of AlcR + analytical Gel
Procedure:
1 µl of Dpn1 (10 U/µl) was added to the reaction, the batch was incubated for one hour at 37°C
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of QuikChange products was added to 150 µl of competent cells and gently mixed.
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellchloramphenicol plate.
0.5 µl of loading buffer were added to 4.5 µl of Dpn1 digested plasmid
Electrophoresis on 1% agarose gel
Lane
1 kbp DNA ladder
QCIII product of AlcR
Results
as expected
[Expand] Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F146+F147 (Ig-Kappa-SigP_SpyTag+Nanoluc) and F145+F42 (pActin+pSB1C3) and negative controls F146 and F42
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103))
Investigator: Jeff
Aim of the experiment: Oligohybridization of mMCSII (mMCSII_fw (O98) + mMCSII_rv (O99)) and (GGGGS)x5_TEV (GGGGSx5_TEV_fw (O102) + GGGGSx5_TEV_rv (O103)).
Procedure:
25 µL of 100 pmol/µl of mMCSII_fw (O98) and mMCSII_rv (O99) were pooled and mixed together/25 µL of 100 pmol/µl of GGGGSx5_TEV_fw (O102) and GGGGSx5_TEV_rv (O103) were pooled and mixed together/
Heating up to 95 °C for 5 min
Cooling at RT in a styropor box overnight
Saturday, June 29th
[Expand] Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)
Investigator: Katrin
Aim of the experiment: Miniprep of Retrafos of P170(SpyTag), P165(t35s), P308(PhyB_36AALinker_C-TEV), P348(Laccase QCV RFC25), P326(Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P361(N-TEV-36AAlinker-PIF6), P364(N-TEV-36AAlinker-PIF3), P266(IgKappa-SigP_Nanoluc)
Procedure:
Miniprep according to QIAGEN QIAprep Kit
[Expand] Transformation of E. coli XL1-blue with ligation product F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F145+F42 (pActin+pSB1C3, ligation overnight at 16°C) and negative control
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)
Investigator: Katrin
Aim of the experiment: Preparative digestion of P402 (Ig-Kappa-SigP_Nanoluc)
Procedure:
reaction batch
volume
reagent
20 µl
P402
4 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF (20 U/µl)
1 µl
SpeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated for 3 h at 37 °C.
[Expand] Preparative gelelectrophoresis of F148, F149 and digestion product of P402
Investigator: Jeff
Aim of the experiment: Preparative gelelectrophoresis of F148, F149 and digestion product of P402.
Procedure:
Preparative gelelectrophoresis was performed on a 1% agarose gel at 90 V for 45 min.
Lane:
1 kbp DNA ladder
F148
F149
Digestion of P402 with AgeI & SpeI
Result:
as expected
as expected
as expected (lower band was cut out)
Gelpurified products were extracted via QIAquick gel extraction kit, QIAGEN.
[Expand] PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA)
Investigator: Jeff
Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin) and P344 (FluA R95KA45IS114T, triple mutant of FluA).
Procedure:
Operational sequence:
Reaction batch with Q5 Mastermix for P143 (pActin amplification):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl
10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl
Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
Reaction batch with Q5 Mastermix for P344 (FluA triple mutant amplification):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O104 (FluA_tripmut_fw)
2.5 µl
10 µM Reverse Primer O105 (FluA_tripmut_rv)
1 µl
Plasmid DNA (P344)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
62 °C
30 s
72 °C
45 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
The PCR program was performed after following scheme for P344 (FluA triple mutant amplification)
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
69 °C
30 s
72 °C
16 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Aim of the experiment: Miniprep of F134 + F135 (SERK-SigP-Laccase + Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F136 + F137 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF6), F136 + F138 (PhyB_36AAlinker_C-TEV_IRES + N-TEV_36AAlinker_PIF3), F143 + F144 (npt-csette + t35S) and ReTraFo of P388 (IgKappa-SigP_SpyTag).
Procedure:
Miniprep according to QIAGEN QIAprep Kit
[Expand] Analytical digestion and gelelectrophoresis of P403 – P418
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P403 – P418.
Procedure:
23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
46 µl
CutSmart Buffer (10x)
5.75 µl
EcoRI-HF (20 U/µl)
5.75 µl
PstI-HF (20 U/µl)
345 µl
ddH2O
=402.5 µl
TOTAL
23x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
17.5 µl
Mastermix for EcoRI & PstI
2.5 µl
Plasmid DNA (P403 – P418)
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2.22 µl of DNA loading buffer (10x) was added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
1000 bp DNA ladder
Digestion of P403 with EcoRI & PstI
Digestion of P404 with EcoRI & PstI
Digestion of P405 with EcoRI & PstI
Digestion of P406 with EcoRI & PstI
Digestion of P407 with EcoRI & PstI
Digestion of P408 with EcoRI & PstI
Digestion of P409 with EcoRI & PstI
Digestion of P410 with EcoRI & PstI
Digestion of P374 with EcoRI & PstI
F153
F154
Result:
corrupt
as expected
as expected
as expected
corrupt
corrupt
corrupt
corrupt
as expected (control)
no PCR product; corrupt
as expected
Lane:
1000 bp DNA ladder
Digestion of P411 with EcoRI & PstI
Digestion of P412 with EcoRI & PstI
Digestion of P246 with EcoRI & PstI (Control)
Digestion of P413 with EcoRI & PstI
Digestion of P414 with EcoRI & PstI
Digestion of P391 with EcoRI & PstI (Control)
Result:
as expected
as expected
as expected
as expected
as expected
as expected
Lane:
1000 bp DNA ladder
Digestion of P415 with EcoRI & PstI
Digestion of P416 with EcoRI & PstI
Digestion of P417 with EcoRI & PstI
Digestion of P418 with EcoRI & PstI
Digestion of P385 with EcoRI & PstI
Result:
as expected
as expected
as expected
as expected
as expected (Control)
[Expand] PCR of P143 (pLuc-Actin, Amplification of pActin)
Investigator: Ingmar
Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin).
Procedure:
Operational sequence:
Reaction batch with Q5 Mastermix for P143 (pActin amplification):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl
10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl
Plasmid DNA (P143)- 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
62 °C
30 s
72 °C
45 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 60 min using NEB 2-Log DNA ladder.
Lane:
1 kbp DNA ladder
1:1000 P143
1:1000 P143
1:10 P143
Result:
length as expected, but low concentration, small amount of side products
length as expected, but low concentration, small amount of side products
length as expected; higher concentration but increased amount of side products, too. Fragment was labelled F155.
Aim of the experiment: Preparative digestion and gelelectrophoresis of P367 (NLS+thermonuclease), P416 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6), P418 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6 ), P404 (t35S_npt-casette), P395 (SpyTag)and F154 (FluA triple mutant).
Procedure:
Batch for preparative digestion of P404 with EcoRI and XbaI, P395 with NgoMIV and SpeI, F154 with XbaI and AgeI, P367 with XbaI and NgoMIV, P416 with SpeI and Pst I and P418 with SpeI and PstI
4 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane
Digestion of P367 with XbaI & NgoMIV
1kb DNA ladder
Digestion of P416 with SpeI & PstI
Digestion of P418 with SpeI & PstI
Results
as expected
as expected
as expected
Lane
Digestion of P404 with EcoRI & XbaI
1kb DNA ladder
Digestion of P395 with NgoMIV & SpeI
Digestion of F154 with XbaI & AgeI
Results
as expected
as expected; lower band was extracted
as expected
Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit
[Expand] Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3)
Investigator: Andi
Aim of the experiment: Ligation of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3), F150 + F160 (SERK-SigP_Nanoluc_SpyTag) and F123 + F161 (FluA triple mutant in pSB1C3).
Procedure:
Ligation batch for F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3):
volume
reagent
2.03 µl
F123 (49.3 ng/µl, 2086 bp)
14.97 µl
F152 (488.2 ng/µl, 102 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F156 + F152 ((GGGGS)x5-TEV-site-linker_NLS_Thermonuclease in pSB1C3):
volume
reagent
0.63 µl
F156 (158.3 ng/µl, 2596 bp)
16.37 µl
F152 (488.2 ng/µl, 102 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):
volume
reagent
0.67 µl
F157 (148.7 ng/µl, 6711 bp)
1.71 µl
F131 (16.4 ng/µl, 627 bp)
14.62 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3):
volume
reagent
0.76 µl
F158 (132.4 ng/µl, 6711 bp)
1.71 µl
F131 (16.4 ng/µl, 627 bp)
14.59 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3):
volume
reagent
1.78 µl
F159 (56.1 ng/µl, 3812 bp)
15.22 µl
F151 (229.4 ng/µl, 37 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F150 + F160 (SERK-SigP_Nanoluc_SpyTag):
volume
reagent
0.59 µl
F150 (169.3 ng/µl, 2660 bp)
16.41 µl
F160 (109 ng/µl, 39 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F123 + F161 (FluA triple mutant in pSB1C3):
volume
reagent
2.03 µl
F123 (49.3 ng/µl, 2086 bp)
2.58 µl
F161 (29.1 ng/µl, 522 bp)
12.39 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Week 11
Monday, July 1st
[Expand] Everyday I'm swaffeling
Investigator: Le swaffleur passive
[Expand] Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter)
Investigator: Ingmar
Aim of the experiment: Preparative digestion and gelelectrophoresis of F155 (pActin PCR product) and P393 (Stress inducible Promoter).
Procedure:
Batch for preparative digestion of F155 (pActin PCR product) with XbaI and PstI
volume
reagent
28.5 µl
F155
4 µl
CutSmart Buffer (10x)
1 µl
XbaI
1 µl
PstI
5.5 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P393 (Stress inducible Promoter)with EcoRI and SpeI"
volume
reagent
20 µl
P393
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
SpeI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Incubation for 2.5 h at 37 °C.
4.5 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches after digestion and they were loaded on a 1% agarose gel
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Lane
1kb DNA ladder
Digestion of F155 with XbaI & PstI
Digestion of P393 with EcoRI & SpeI
Results
as expected, the upper band was cut out
as expected, the lowest band was cut out
Extracted via QIAGEN gel extraction kit, digested PCR product purified via PCR Purification Kit
[Expand] Ligation of F162 (pActin) + F42(pSB1C3 backbone)
Investigator: Ingmar
Aim of the experiment: Ligation of F162 (pActin) + F42(pSB1C3 backbone)
Procedure:
Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)
volume
reagent
1.36 µl
F42 (100 ng)
6.64 µl
F162
91 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (NEB)
=20 µl
TOTAL
Negative controls were prepared by replacing the volume of the insert by ddH2O.
The ligation was performed for 1 hour at room temperature; the ligation batch was transferred to 16 °C afterwads over night.
[Expand] Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature
Investigator: Ingmar
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation for 1 h at room temperature
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
Tuesday, July 2nd
[Expand] Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C
Investigator: Ingmar
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation over night at 16 °C.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Picking of F123 + F152, F157 + F131, F158 + F131, F159 + F151 and F123 + F161
Investigator: Louise
Aim of the experiment: Picking of F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3).
Procedure:
Picked colonies were transfered into 5 ml LB-medium with 5 µl of Chloramphenicol and incubated for at 37 °C in the 180 rpm cell-culture shaker.
[Expand] Ligation of F162 (pActin) + F42(pSB1C3 backbone) and of F163 (stress inducible Promoter + RFP ) + F159 (t35S-NPT-pSB1C3)
Investigator: Ingmar
Aim of the experiment: As the ligation of the prmoter done on Monday failed, it is repeated with an decreased overall volume resulting in an increased DNA concentration: Ligation of F162 (pActin) + F42(pSB1C3 backbone). Furthermore the stress inducible Promoter cloned to RFP (F163) should be ligated into a pSB1C3 in front og t35S and NPT.
Procedure:
Ligation batch for ligation of F162 (pActin) + F42(pSB1C3 backbone)
volume
reagent
1.36 µl
F42 (100 ng)
6.64 µl
F162
1 µl
T4 ligase buffer (10x)
1 µl
T4 ligase
=10 µl
TOTAL
The reaction was prepared twice: one time using a ligase provided by NEB and one time using a ligase providied by Thermo Scientific. Negative controls were prepared by replacing the volume of the insert by ddH2O.
Ligation batch for ligation of F163 (stress inducible promoter + RFP) + F159(t35S-NPT-pSB1C3 backbone)
volume
reagent
1.79 µl
F159 (100 ng)
8.61 µl
F163
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase
6,6 µl
H2O
=20 µl
TOTAL
The ligation was performed for 2 hours at room temperature; the ligation batch was transferred to 16 °C afterwads over night.
[Expand] Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3)
Investigator: Ingmar
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F162+F42 (pActin+pSB1C3, ligation batch volume 10 µl, reaction performed at room temperature) and with F159+F163 (stress inducible promoter+RFP+t35S+NPT in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] PCR of P143 (pLuc-Actin, Amplification of pActin)
Investigator: Ingmar
Aim of the experiment: PCR of P143 (pLuc-Actin, Amplification of pActin). The PCR product was phosphorylated afterwards to test blunt end cloning into a vector.
Procedure:
Operational sequence:
Reaction batch for PCR with Q5 Mastermix for P143 (pActin amplification):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O96 (pActin_Oryza_fw)
2.5 µl
10 µM Reverse Primer O97 (pActin_Oryza_rv)
1 µl
Plasmid DNA (P143)- 1:10 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme for P143 (pActin amplification):
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
62 °C
30 s
72 °C
45 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
The PCR was performed in two different cyclers in parallel to elucidate wheather this might have an influence on the product. Furthermore one sample contained a 100 fold increased template concentration.
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
Reaction batch for phosphorylation of thePCR product:
volume
reagent
15 µl
PCR product
2 µl
10 x forward buffer A
2 µl
10 mM ATP
1 µl
Polynucleotide Kinase (Thermo Scientific)
=20 µL
TOTAL
The reaction was performed for 1 h at 37 °C. Afterwards the mixture was inactivated by heat for 10 min at 75 °C.
[Expand] Quickchange mutagenesis of P143 (Actin promoter)
Investigator: Ingmar
Aim of the experiment: Deliting the forbidden EcoRI restriction site in the promoter.
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid P143 template
0.5 µl
1:10 dilution of O106
19. 5 µl
ddH2O
1 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid P143 template
0.5 µl
O107
19.5 µl
ddH2O
1 µl
dNTP mix
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 sec
2
15
95°C
30 sec
55°C
1 min
67°C
6,5 min
Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied. Furthermore 1 µl Pfu Ultra II DNA polymerase was added.
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag)
Investigator: Louise, Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) and P395 (SpyTag).
Batch for preparative digestion of P352 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):
volume
reagent
20 µl
P352
4 µl
CutSmart Buffer (10x)
1 µl
SpeI
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P358 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1):
volume
reagent
20 µl
P358
4 µl
CutSmart Buffer (10x)
1 µl
SpeI
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P395 (SpyTag):
volume
reagent
20 µl
P395
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV
1 µl
SpeI
14 µl
ddH2O
=40 µl
TOTAL
Incubation at 37 °C; 2.5 h
4.44 µl of DNA loading buffer (10x) were added to the 40 µl reaction batches, digested with NgoMIV & SpeI (P395), after digestion and they were loaded on a 1% agarose gel.
Preparative gelelectrophoresis was performed at 90 V for 60 min.
Reaction batches which were digested with SpeI & PstI were purified with QIAquick PCR purification kit, QIAGEN.
Lane
1kb DNA ladder
Digestion of P395 with NgoMIV & SpeI
Results
as expected; lower band was cut out
Extracted via QIAquick gel extraction kit, QIAGEN.
[Expand] Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES) and F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)
Investigator: Jeff
Aim of the experiment: Ligation of F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).
Procedure:
Ligation batch for F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:
volume
reagent
1.22 µl
F164 (81.7 ng/µl, 3524 bp)
3.25 µl
F131 (16.4 ng/µl, 627 bp)
12.53 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), vector:insert = 1:3:
volume
reagent
1.65 µl
F165 (60.7 ng/µl, 3224 bp)
3.56 µl
F131 (16.4 ng/µl, 627 bp)
11.79 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at RT for 1 h.
[Expand] Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F164 + F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3+ IRES), F165 + F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES) and retransformations of P352, P358 and P395.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)
Investigator: Jeff
Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).
Procedure:
Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:
volume
reagent
0.59 µl
F150 (169.3 ng/µl, 2660 bp)
2.67 µl
F166 (3.3 ng/µl, 39 bp)
13.79 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 16 °C overnight.
[Expand] Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)
Investigator: Ingmar, Jeff
Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F123 + F152 ((GGGGS)x5-TEV-site-linker in pSB1C3), F157 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F158 + F131 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3/6_IRES in pSB1C3), F159 + F151 (mMCSII_t35S_npt-casette in pSB1C3) and F123 + F161 (FluA triple mutant in pSB1C3)
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
The resulting tubes were labeled as P420–P432.
[Expand] Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)
Investigator: Jeff
Aim of the experiment: Picking of of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).
Procedure:
Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight
Wednesday, July 3rd
[Expand] Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES)
Investigator: Ingmar
Aim of the experiment: Picking of of E. coli XL1 blue transformed with F162 + F42 (pActin + pSB1C3), F163 + F159 (Stress inducible promotor_RFP + t35S_NPT in pSB1C3, P395, P352, P358, F164+F131 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES), F165+F131 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in pSB1C3 + IRES).
Procedure:
Picked colonies tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 5 mL of LB-medium + 5 µL chloramphenicol(1000x).
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.
[Expand] Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3)
Investigator: Jeff
Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F152 + F156 ((GGGGS)x5-TEV-site-linker + NLS_Thermonuclease in pSB1C3).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
The resulting tubes were labeled as P433 – P435.
[Expand] Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Analytical digestion and gelelectrophoresis of P420 – P435
Investigator: Ingmar
Aim of the experiment: Analytical digestion and gelelectrophoresis of P420 – P435.
Procedure:
Lane:
1000 bp DNA ladder
Digestion of P420 with EcoRI & PstI
Digestion of P421 with EcoRI & PstI
Digestion of F123 with EcoRI & PstI
Digestion of P422 with EcoRI & PstI
Digestion of P429 with EcoRI & PstI
Digestion of P430 with EcoRI & PstI
Digestion of P431 with EcoRI & PstI
Digestion of P432 with EcoRI & PstI
1000 bp DNA ladder
Result:
as expected
as expected
as expected
corrupt
corrupt
corrupt
corrupt
Lane:
1000 bp DNA ladder
Digestion of P424 with EcoRI & PstI
Digestion of P425 with EcoRI & PstI
Digestion of P426 with EcoRI & PstI
Digestion of P427 with EcoRI & PstI
Digestion of P428 with EcoRI & PstI
Digestion of P404 with EcoRI & PstI
Digestion of P433 with EcoRI & PstI
Digestion of P434 with EcoRI & PstI
Digestion of P435 with EcoRI & PstI
Digestion of P367 with EcoRI & PstI
1000 bp DNA ladder
Result:
as expected
as expected
as expected
corrupt
corrupt
corrupt
corrupt
as expected (control)
no PCR product; corrupt
as expected
Thursday, July 4th
[Expand] Quickchange mutagenesis I of P444 (Actin promoter)
Investigator: Louise
Aim of the experiment: Deleting the forbidden EcoRI restriction site in the promoter.
Procedure:PCR
Reaction batch 1
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid P444 template
1.0 µl
1:10 dilution of O106
1.0 µl
1:10 dilution of O107
18.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U / µl)
Reaction batch 2
volume
reagent
12.5 µl
Q5 Polymerase Mastermix
1 µl
Plasmid P444 template
1.25 µl
1:10 dilution of O106
1.25 µl
1:10 dilution of O107
8.0 µl
ddH2O
1 µl
dNTP mix 50 mM
PCR cycling parameters for batch 1
Cycles
Temperature
Time
1
95 °C
30 sec
20
95°C
30 sec
52°C
1 min
68°C
5 min
4°C
HOLD
PCR cycling parameters for batch 2
Cycles
Temperature
Time
1
98 °C
30 sec
20
98°C
10 sec
55°C
30 sec
72°C
4 min
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
The successful amplification was verified on an agerose gel and the PCR-product was transformed (by Jeff)
[Expand] Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150
Investigator: Jeff, Katrin
Aim of the experiment: Ligation of F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag) and negative control F150
Procedure:
Ligation batch for F150 + F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), vector:insert = 1:6:
volume
reagent
0.59 µl
F150 (169.3 ng/µl, 2660 bp)
2.67 µl
F166 (3.3 ng/µl, 39 bp)
13.79 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of insert F166
[Expand] Test digestion of pAct in pSB1C3 (p435 to p448)
Investigator: Jeff
App. 500 ng of DNA from 13 different clones were digeted with EcoRI and PstI
Mastermix consisting of 3 µl of each enzyme 30 µl Cutsmart buffer and 120 µl water
p444 and p447 were sent for sequencing
[Expand] Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls
Investigator: Katrin
Aim of the experiment: Transformation of E. coli XL1-blue with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher) and negative controls
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Preparative digestion of P434, P412, P462, P453
Investigator: Katrin
Aim of the experiment: Preparative digestion of P434, P412, P462, P453
Batch for preparative digestion of P434
volume
reagent
20 µl
P434
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P412
volume
reagent
20 µl
P412
4 µl
CutSmart Buffer (10x)
1 µl
XbaI
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P462
volume
reagent
20 µl
P462
4 µl
CutSmart Buffer (10x)
1 µl
PstI-HF
1 µl
SpeI
14 µl
ddH2O
=40 µl
TOTAL
Batch for preparative digestion of P453
volume
reagent
20 µl
P453
4 µl
CutSmart Buffer (10x)
1 µl
PstI-HF
1 µl
SpeI
14 µl
ddH2O
=40 µl
TOTAL
Incubation at 37 °C; 3 h, then put into freezer
[Expand] Ligation of F142+F147 (IgK-SigP_Spycatcher + NanoLuc) and F141+F139 (SpyCatcher + IgK-SigP_NanoLuc)
Investigator: Louise
Aim of the experiment: Ligation of F142+F147 and F139+F141
Procedure:
Ligation batch for F142+F147:
volume
reagent
0.81 µl
F142 (123.4 ng/µl, 2489 bp)
3.51 µl
F147 (17.5 ng/µl, 510 bp)
12.68 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F141+F139
volume
reagent
1.83 µl
F141 (54.6 ng/µl, 2425 bp)
3.43 µl
F139 (21.0 ng/µl, 583 bp)
11.74 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of inserts F147 and F139
Friday, July 5th
[Expand] Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls
Investigator: Katrin
Aim of the experiment: Ligation of F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls
Procedure:
Ligation batch for F122+F169
volume
reagent
1.99 µl
F122 (50.3 ng/µl, 2369 bp)
4.32 µl
F169 (18.1 ng/µl, 618 bp)
10.69 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F170+F168
volume
reagent
2.01 µl
F170 (49.7 ng/µl, 4168 bp)
4.11 µl
F168 (11.0 ng/µl, 628 bp)
10.88 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 22 °C for 1h
negative control was prepaed with water instead of inserts F169 and F168
[Expand] Transformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls
Investigator: Katrin
Aim of the experiment: TTransformation of E. coli XL1-blue with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_Nanoluc) and negative controls
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)
Investigator: Katrin
Aim of the experiment: Picking of E. coli XL1 blue transformed with ligation product F150+F166 (IgKappa-SigP_NanoLuc in pSB1C3 + SpyTag), F142+F147 (Ig-Kappa-SigP_SpyCatcher + Nanoluc), F139+F141 (Ig-Kappa-SigP_Nanoluc + SpyCatcher)
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
2 colonies were picked for F150+F166 and F139+F141, 8 colonies were picked for F142+F147 (many colonies on negatibe controls)
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overday.
[Expand] Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B)
Investigator: Rosario
Aim of the experiment: Sequencing of P3 (pTEF2 in pTUM100), P341 (QCI of AlcR), P453 (SERK-SigP_SpyCatcher_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P462 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 + IRES in pSB1C3), P300 (Phytotchrome B).
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
Barcode3
Barcode4
P3
FR02233147 (O5)
P341
FR02233146 (O3)
FR02233145 (O4)
FR02233144 (O100)
FR02233143 (O101)
P453
FR02233142 (O4)
P462
FR02233141 (O4)
P300
FR02233140 (O3)
FR02233139 (O4)
FR02233138 (O110)
FR02233137 (O111)
[Expand] Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site
Investigator: Jeff
Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.
Procedure:
Reaction batch 1:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid P447 template (1:100)
1.0 µl
1:10 dilution of O106 (=10 µM)
1.0 µl
1:10 dilution of O107 (=10 µM)
18.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
Reaction batch 2:
volume
reagent
25 µl
Q5 Polymerase Mastermix
1 µl
Plasmid P447 template (1:100)
2.5 µl
1:10 dilution of O106 (=10 µM)
2.5 µl
1:10 dilution of O107 (=10 µM)
8.0 µl
ddH2O
1 µl
dNTP mix 50 mM
PCR cycling parameters for batch 1
Cycles
Temperature
Time
1
95 °C
30 sec
20
95°C
30 sec
52°C
1 min
68°C
4 min
1
4°C
HOLD
PCR cycling parameters for batch 2
Cycles
Temperature
Time
1
98 °C
30 sec
20
98°C
10 sec
55°C
30 sec
72°C
2 min
3
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] QuikChange of pYes (P3)
Investigator: Rosario
Aim of the experiment: QuikChange of pYes (P3)
Reaction batch
volume
reagent
Additional information
2.5 µl
10x Pfu Ultra II buffer
1 µl
Plasmid template (P3), (1:10 diluted)
need to get between 2.5 ng and 25 ng for a reaction batch of 25 µl
1 µl
1:10 dilution of O5 (=10 µM)
19 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
PCR cycling parameters
Segment
Cycles
Temperature
Time
1
1
95 °C
30 s
2
20
95 °C
30 s
52 °C
1 min
68 °C
4 min
3
4 °C
hold
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100)
Investigator: Jeff
Procedure: Analytical gelelectrophoresis of the DpnI-digested QuikChange products of P447 (pActin in pSB1C3) and P3 (pTEF2 in pTUM100) to check whether QuikChange might be worked.
Procedure:
4.5 µl of the QuikChange products of P447 (PfuUltraII Polymerase batch and Q5 Polymerase batch (see above)) and P3 (pTEF2 in pTUM100) were mixed together 0.5 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed on a 1% agarose gel at 90 V for 40 min.
Lane:
2 log DNA ladder
QC of P3 with O44 & O45 (PfuUltraII)
QC of P447 with O106 & O107 (PfuUltraII)
QC of P447 with O106 & O107 (Q5)
Result:
as expected
no product visible
no product visible
[Expand] Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100)
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (PfuUltraII and Q5) and of P3 (pTEF2 in pTUM100) (see above).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
Saturday, July 6th
[Expand] Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site
Investigator: Le swaffeleur
Aim of the experiment: Quikchange mutagenesis of P447 (Actin promoter) to remove forbidden EcoRI restriction site.
Procedure:
Reaction batch 1:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P447 template (1:100)
1.0 µl
1:10 dilution of O106 (=10 µM)
16.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl
TOTAL
Reaction batch 2:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P447 template (1:100)
1.0 µl
1:10 dilution of O107 (=10 µM)
16.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl
TOTAL
The two batches were cycled seperately for the first 10 rounds.
PCR cycling parameters
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
54°C
1 min
68°C
4 min
1
4°C
HOLD
After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:
PCR cycling parameters after poooling the reaction batches together:
Cycles
Temperature
Time
1
98 °C
30 sec
20
98°C
10 sec
54°C
30 sec
72°C
2 min
3
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3)
Investigator: Jeff
Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P447 (pActin in pSB1C3).
Procedure:
9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 30 min on a 1% agarose gel.
Lane:
2 log DNA ladder
QC of P447 with O106 & O107 (PfuUltraII, without DMSO)
QC of P447 with O106 & O107 (PfuUltraII, with DMSO)
Result:
as expected
as expected
[Expand] Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3)
Investigator: Jeff, Le Swaffeleur
Aim of the experiment: Transformation of E. coli XL1-blue with QuikChange products of P447 (pActin in pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
8 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
[Expand] Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)
Investigator: Jeff
Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation product F122+F169 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA) and F170+F168 (SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
2 colonies were picked for F122+F169 and F170+F168.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Sunday, July 7th
[Expand] Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc)
Investigator: Jeff, Katrin
Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F170+F168(SERK-SigP_SpyCatcher_Strep-TagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES + Ig-Kappa-SigP_SpyTag_NanoLuc).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
The resulting tubes were labeled as P475 and P476.
[Expand] Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)
Investigator: Jeff, Katrin
Aim of the experiment: Analytical digestion and gelelectrophoresis of P464 – P476 and P402 (control)
16x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
17.5 µl
Mastermix for EcoRI & PstI
2.5 µl
Plasmid DNA (P464 – P476, P402)
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
1000 bp DNA ladder
Digestion of P464 with EcoRI & PstI
Digestion of P465 with EcoRI & PstI
Digestion of P466 with EcoRI & PstI
Digestion of P467 with EcoRI & PstI
Digestion of P402 with EcoRI & PstI (control)
Digestion of P475 with EcoRI & PstI
Digestion of P476 with EcoRI & PstI
Result:
as expected
as expected
as expected
as expected
as expected
as expected
as expected
Lane:
1000 bp DNA ladder
Digestion of P468 with EcoRI & PstI
Digestion of P469 with EcoRI & PstI
Digestion of P470 with EcoRI & PstI
Digestion of P471 with EcoRI & PstI
Digestion of P472 with EcoRI & PstI
Digestion of P473 with EcoRI & PstI
Digestion of P474 with EcoRI & PstI
Result:
corrupt
corrupt
corrupt
corrupt
corrupt
corrupt
corrupt
[Expand] PCR of P378 (Proteinphosphotase 1)
Investigator: Katrin, Jeff
Aim of the experiment: PCR of P378 (Proteinphosphotase 1).
Procedure:
Reaction batch for PCR with Q5 Mastermix for P378 (Proteinphosphotase 1):
volume
reagent
25 µl
2x Q5 Master Mix
1 µl
dNTP mix (normally not necessary!)
2.5 µl
10 µM Forward Primer O112 (PP1 for)
2.5 µl
10 µM Reverse Primer O113 (PP1 rev)
1 µl
Plasmid DNA (P378) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme for P378(PP1 amplification):
Cycling parameters:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
62 °C
30 s
72 °C
30 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting product was labeled as F174.
[Expand] Analytical gelelectrophoresis of F174
Investigator: Jeff
Aim of the experiment: Analytical gelelectrophoresis of F174.
Procedure:
9 µl of the PCR product F174 (=PCR of P378 with O112/O113) products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
Lane:
2 log DNA ladder
F174
Result:
as expected
[Expand] Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag)
Investigator: Katrin, Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P422 (FluA triple mutant), P464 (IgKappa-SigP_NanoLuc_SpyCatcher) and P466 (IgKappa-SigP_NanoLuc_SpyTag).
Procedure:
Reaction batch for P422 (FluA triple mutant):
volume
reagent
20 µl
P422
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV
1 µl
SpeI
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P464 (IgKappa-SigP_NanoLuc_SpyCatcher):
volume
reagent
20 µl
P464
4 µl
CutSmart Buffer (10x)
1 µl
SpeI
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P466 (IgKappa-SigP_NanoLuc_SpyTag):
volume
reagent
20 µl
P466
4 µl
CutSmart Buffer (10x)
1 µl
SpeI
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P422 with NgoMIV & SpeI
2 log DNA ladder
Digestion of P464 with XbaI & PstI
Digestion of P466 with XbaI & PstI
Results
as expected; lower band was cut out
as expected; lower band was cut out
as expected; lower band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1)
Investigator: Katrin, Jeff
Aim of the experiment: Preparative digestion of P379 (IgKappa-SigP_SpyCatcher) and F174 (Proteinphosphotase 1).
Procedure:
Reaction batch for P379 (IgKappa-SigP_SpyCatcher):
volume
reagent
20 µl
P379
4 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF
1 µl
PstI-HF
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for F174 (Proteinphosphotase 1):
volume
reagent
25 µl
F174
5 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF
1 µl
PstI-HF
18 µl
ddH2O
=50 µl
TOTAL
Reaction batches were incubated at 37 °C for 4 h.
After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.
[Expand] Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)
Investigator: Jeff
Aim of the experiment: Ligation of F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).
Procedure:
Ligation batch for F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES
in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), vector:insert = 1:3:
volume
reagent
2.01 µl
F170 (49.7 ng/µl, 4168 bp)
3.50 µl
F179 (12.9 ng/µl, 628 bp)
11.49 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), vector:insert = 1:3:
volume
reagent
1.65 µl
F171 (60.05 ng/µl, 3868 bp)
5.80 µl
F178 (12.4 ng/µl, 928 bp)
9.55 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), vector:insert = 1:3:
volume
reagent
0.61 µl
F176 (163.9 ng/µl, 2489 bp)
3.51 µl
F147 (17.5 ng/µl, 510 bp)
12.88 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), vector:insert = 1:3:
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2117 bp)
2.90 µl
F177 (25.5 ng/µl, 522 bp)
13.18 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F123+F175 (pSB1C3 only + PP1), vector:insert = 1:3:
volume
reagent
0.92 µl
F123 (49.3 ng/µl, 2086 bp)
2.90 µl
F175 (12.3 ng/µl, 972 bp)
3.59 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 22 °C for 1 h
Negative controls were also prepared with water instead of insert.
[Expand] Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-blue with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker)
Investigator: Jeff
Aim of the experiment: Picking of E. coli XL1 blue transformed with QuikChange products of P447 (pActin) and ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
8 colonies were picked for QuikChange products of P447 (pActin)and F139+F141 and 4 colonies were picked for ligation product F123+F152 ((GGGGS)x5-TEV-cleavage-site-linker).
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Week 12
Monday, July 8th
[Expand] Analytical digestion and gelelectrophoresis of P477-P484 (pActin)
Investigator: Rosario, Ingmar
Aim of the experiment: Analytical digestion and gelelectrophoresis of P477-P484 (pActin in pSB1C3) after QC of forbidden EcoRI Restriction site.
10x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
15 µl
Cut Smart buffer
2 µl
EcoRI
2 µl
PstI
30 µl
ddH2O
=49 µl
TOTAL
The volume of plasmid DNA added was calculated in order to have equal amounts of DNA of approximately 550 ng
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
1000 bp DNA ladder
Digestion of P477 with EcoRI & PstI
Digestion of P478 with EcoRI & PstI
Digestion of P479 with EcoRI & PstI
Digestion of P480 with EcoRI & PstI
Digestion of P481 with EcoRI & PstI (control)
Digestion of P482 with EcoRI & PstI
Digestion of P483 with EcoRI & PstI
Digestion of P484 with EcoRI & PstI
Result:
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
all quickchanges were successfull.
[Expand] Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion
Investigator: Rosario, Ingmar
Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 50 bp deletion.
Procedure:
Reaction batch 1:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
2,67 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O108 (=10 µM)
17.330 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl
TOTAL
Reaction batch 2:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
2.67 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O109 (=10 µM)
17.33 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl
TOTAL
The two batches were cycled seperately for the first 10 rounds.
PCR cycling parameters
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
54°C
1 min
72°C
4.5 min
1
4°C
HOLD
After the ten cycles the two reaction batches were pooled together and were additionally cycled for 20 rounds with the following conditions:
PCR cycling parameters after poooling the reaction batches together:
Cycles
Temperature
Time
1
98 °C
30 sec
20
98°C
10 sec
54°C
30 sec
72°C
2 min
3
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
[Expand] Sequencing of P477, P486, P487, P464, P466, P476, P435
Investigator: Rosario
Aim of the experiment: Sequencing of P477 (Actin promoter), P486, P487, P464, P466, P476, P435.
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
P477
FR02233136 (O3)
FR02233135 (O4)
P486
FR02233134 (O3)
P487
FR02233133 (O3)
P464
FR02233132 (O4)
P466
FR02233131 (O4)
P476
FR02233130 (O3)
FR02233129 (O4)
P435
FR02233128 (O3)
[Expand] Preparative digestion and gelelectrophoresis of P187 (LMU GFP)
Investigator: Rosario
Aim of the experiment: Preparative digestion and gelelectrophoresis of P187 (LMU GFP).
Procedure:
Reaction batch for P187 :
volume
reagent
20 µl
P187
4 µl
CutSmart Buffer (10x)
1 µl
NgoMIV
1 µl
SpeI
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch was incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P187 with NgoMIV & SpeI
2 log DNA ladder
Results
as expected; lower band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP)
Investigator: Rosario
Aim of the experiment: Ligation of F109+F180 (SERK-SigP in pSB1C3 + LMU GFP) and F92+F180 (IgKappa in pSB1C3 + LMU GFP).
Procedure:
Ligation batch for F109+F180 (SERK-SigP in pSB1C3 + LMU GFP), vector:insert = 1:3:
volume
reagent
4.1 µl
F180 (726 bp)
0.9 µl
F179 (2177 bp)
12 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F92+F180 (IgKappa in pSB1C3 + LMU GFP), vector:insert = 1:3:
volume
reagent
4.15 µl
F180 (726 bp)
0.85 µl
F92 (2144 bp)
12 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 22 °C for 1 h
Negative controls were also prepared with water instead of insert.
[Expand] Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1))
Investigator: Rosario
Aim of the experiment: Picking of E. coli XL1-blue transformed with ligation products F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher), F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) and F123+F175 (pSB1C3 only + PP1)).
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
2 colonies were picked for all ligation products except F176+F147 for which 4 colonies were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Tuesday, July 9th
[Expand] Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag)
Investigator: Jeff, Florian
Aim of the experiment: Miniprep of E. coli XL1 blue transformed with F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc), F123+F175 (pSB1C3 only + PP1), F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant), F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) and F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN).
The resulting tubes were labeled as P488 to P499.
[Expand] Analytical digestion and gelelectrophoresis of P488-P499 (several ligations)
Investigator: Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of P488-P499 (several ligations).
15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
30 µl
Cut Smart buffer
3.75 µl
EcoRI
3.75 µl
PstI
225 µl
ddH2O
=262,5 µl
TOTAL
17.5 µl master mix and 2.5 µl plasmid DNA were mixed.
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
1 kb DNA ladder
Digestion of P488 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI
Digestion of P489 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI
Digestion of P490 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI
Digestion of P491 F176+F147 (IgKappa-SigP_SpyCatcher in pSB1C3 + NanoLuc) with EcoRI & PstI
Digestion of P492 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI
Digestion of P493 F123+F175 (pSB1C3 only + PP1) with EcoRI & PstI
Result:
as expected (950 bp)
as expected (950 bp)
as expected (950 bp)
as expected (950 bp)
as expected (972 bp)
as expected (972 bp)
Lane:
1 kb DNA ladder
Digestion of P422 F123+F161 (FluA triple mutant + pSB1C3) with EcoRI & PstI (control)
Digestion of P494 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI
Digestion of P495 F109+F177 (SERK-SigP in pSB1C3 + FluA triple mutant) with EcoRI & PstI
Digestion of P496 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI
Digestion of P497 F171+F178 (SERK-SigP_SpyTag_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyCatcher) with EcoRI & PstI
Digestion of P498 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI
Digestion of P499 F170+F179 (SERK-SigP_SpyCatcher_StrepTagII-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_NanoLuc_SpyTag) with EcoRI & PstI
Result:
as expected (522 bp)
strange
as expected (622 bp)
roughly as expected (2768 bp)
roughly as expected (2768 bp)
roughly as expected (2768 bp)
roughly as expected (2768 bp)
[Expand] Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion
Investigator: Florian
Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 50 bp deletion.
Procedure:
9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
Lane:
1 kb DNA ladder
QC of P477
Result:
no product
[Expand] Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion, second attempt
Investigator: Jeff
Aim of the experiment: Quikchange mutagenesis of P477 (Actin promoter) to insert 60 bp deletion.
Procedure:
Reaction batch 1:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
3 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O108 (=10 µM)
17.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=25 µl
TOTAL
Reaction batch 2:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
3.0 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O109 (=10 µM)
17.0 µl
ddH2O
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
=20 µl
TOTAL
The two batches were cycled seperately for the first 25 rounds.
PCR cycling parameters
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
52°C
1 min
72°C
4.5 min
1
4°C
HOLD
After the ten cycles the two reaction batches were pooled together, 0.5 µl of polymerase and 1 µl of dNTP-mix was added and the reaction was cycled for 25 rounds with the following conditions:
PCR cycling parameters after poooling the reaction batches together:
Cycles
Temperature
Time
1
98 °C
30 sec
20
98°C
10 sec
52°C
30 sec
72°C
2 min
3
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.
Wednesday, July 10th
[Expand] Preparative digestion of P495 (SERK-SigP_FluA)
Investigator: Jeff
Aim of the experiment: Preparative digestion of P495 (SERK-SigP_FluA).
Procedure:
Reaction batch for P495 (SERK-SigP_FluA):
volume
reagent
20 µl
P495
4 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
After digestion, linearized DNA was purified with QIAquick PCR purification kit, QIAGEN.
[Expand] Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1)
Investigator: Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P486 ((GGGGS)x5-TEV-site-linker), P488 (IgKappa-SigP_SpyCatcher_NanoLuc) and P492 (Proteinphosphotase 1).
Procedure:
Reaction batch for P486 ((GGGGS)x5-TEV-site-linker):
volume
reagent
20 µl
P486
4 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
AgeI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P488 (IgKappa-SigP_SpyCatcher_NanoLuc):
volume
reagent
20 µl
P488
4 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P492 (Proteinphosphotase 1)):
volume
reagent
20 µl
P492
4 µl
CutSmart Buffer (10x)
2 µl
NgoMIV (10 U/µl)
2 µl
SpeI (10 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P486 with XbaI & AgeI
2 log DNA ladder
Digestion of P488 with XbaI & PstI
Digestion of P492 with NgoMIV & SpeI
Results
as expected; lower band was cut out
as expected; lower band was cut out
as expected; lower band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)
Investigator: Jeff
Aim of the experiment: Ligation of F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).
Procedure:
Ligation batch for F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), vector:insert = 1:3:
volume
reagent
0.63 µl
F156 (158.3 ng/µl, 2596 bp)
2.88 µl
F182 (4.1 ng/µl, 102 bp)
13.49 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:
volume
reagent
0.54 µl
F181 (185.7 ng/µl, 2705 bp)
6.21 µl
F135 (17.8 ng/µl, 996 bp)
10.25 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), vector:insert = 1:3:
volume
reagent
1.65 µl
F171 (60.5 ng/µl, 3859 bp)
1.22 µl
F183 (59 ng/µl, 928 bp)
14.13 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1), vector:insert = 1:3:
volume
reagent
0.92 µl
F109 (108.2 ng/µl, 2117 bp)
2.44 µl
F184 (54.9 ng/µl, 972 bp)
13.64 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 16 °C overnight.
Negative controls were also prepared with water instead of insert.
Thursday, July 11th
[Expand] Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498
Investigator: Jeff
Aim of the experiment: Sequencing of P294, P296, P342, P488, P492, P495, P496 and P498.
Procedure:
The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
Barcode3
P294
FR02233072 (O100)
P296
FR02233073 (O3)
FR02233074 (O4)
FR02233075 (O100)
P342
FR02233076 (O3)
FR02233077 (O4)
FR02233078 (O100)
P488
FR02233079 (O4)
P492
FR02233080 (O3)
FR02233081 (O4)
P495
FR02233082 (O3)
P496
FR02233083 (O3)
FR02233084 (O4)
P498
FR02233085 (O3)
FR02233086 (O4)
Friday, July 12th
[Expand] QuikChange of P477 (pActin) to insert missing 60 bp
Investigator: Jeff
Aim of the experiment: QuikChange of P477 (pActin) to insert missing 60 bp.
Procedure:
Reaction batch #1, with 5% DMSO and O108 as oligonucleotide:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O108 (=10 µM)
1 µl
dNTP mix 50 mM
1.25 µl
DMSO
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl
ddH2O
=25 µl
TOTAL
Reaction batch #2, with 5% DMSO and O109 as oligonucleotide:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O109 (=10 µM)
1 µl
dNTP mix 50 mM
1.25 µl
DMSO
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
14.75 µl
ddH2O
=25 µl
TOTAL
Reaction batch #3, O108 as oligonucleotide:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O108 (=10 µM)
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl
ddH2O
=25 µl
TOTAL
Reaction batch #4, O109 as oligonucleotide:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P477 template (1:100)
1.0 µl
1:10 dilution of O109 (=10 µM)
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
16 µl
ddH2O
=25 µl
TOTAL
Every reaction batch type was created twice; one batch of each type was used for the program with 55 °C as annealing temperature and one was used with 65 °C as annealing temperature.
The batches were cycled seperately for the first 10 rounds
PCR cycling parameters with 55 °C annealing temperature:
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
55°C
1 min
72°C
4.5 min
1
4°C
HOLD
PCR cycling parameters with 65 °C annealing temperature:
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
65°C
1 min
72°C
4.5 min
1
4°C
HOLD
After the ten cycles the reaction batches (#1 with #2 and #3 with #4) were pooled together and the reaction was cycled for further 20 rounds with the following conditions:
PCR cycling parameters with 55 °C annealing temperature:
Cycles
Temperature
Time
1
95 °C
30 sec
20
95°C
30 sec
55°C
1 min
72°C
4.5 min
1
4°C
HOLD
PCR cycling parameters with 65 °C annealing temperature:
Cycles
Temperature
Time
1
95 °C
30 sec
20
95°C
30 sec
65°C
1 min
72°C
4.5 min
1
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.
[Expand] Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion
Investigator: Jeff
Aim of the experiment: Analytical gelelectrophoresis of QuikChange products of P477 (Actin promoter) to insert 60 bp deletion.
Procedure:
9 µl of the QuikChange products were mixed with 1 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 100 V for 40 min on a 1% agarose gel.
Lane:
2 log DNA ladder
QC of P477 with DMSO and annealing temperature at 55 °C
QC of P477 with DMSO and annealing temperature at 65 °C
QC of P477 with annealing temperature at 55 °C
QC of P477 with annealing temperature at 65 °C
Result:
no product
no product
no product
no product
[Expand] Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)
Investigator: Jeff
Aim of the experiment: Picking of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
2 colonies were picked for F156+F182, F171+F182, F109+F184 and 6 colonies were picked for ligation product F181+F135.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Saturday, July 13th
[Expand] Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1)
Investigator: Jeff
Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products F156+F182 (NLS_NucA in pSB1C3 + (GGGGS)x5-TEV-site-linker), F181+F135 (SERK-SigP_FluA in pSB1C3 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), F171+F183 (SERK-SigP_SpyTag_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES in pSB1C3 + IgKappa-SigP_SpyCatcher_NanoLuc), F109+F184 (SERK-SigP in pSB1C3 + Proteinphosphotase 1).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P508 to P519.
[Expand] Analytical digestion and gelelectrophoresis of P508 – P519
Investigator: Le Swaffeleur
Aim of the experiment: Analytical digestion and gelelectrophoresis of P508 – P519.
Procedure:
15x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
26 µl
Cut Smart buffer
3.25 µl
EcoRI-HF (20 U/µl)
3.25 µl
PstI-HF (20 U/µl)
195 µl
ddH2O
=227.5 µl
TOTAL
17.5 µl master mix and 2.5 µl plasmid DNA (P508 – P519) were mixed.
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2 log DNA ladder
P508, digested with EcoRI & PstI
P509, digested with EcoRI & PstI
P510, digested with EcoRI & PstI
P511, digested with EcoRI & PstI
P512, digested with EcoRI & PstI
P513, digested with EcoRI & PstI
P514, digested with EcoRI & PstI
P515, digested with EcoRI & PstI
P516, digested with EcoRI & PstI
P517, digested with EcoRI & PstI
P518, digested with EcoRI & PstI
P519, digested with EcoRI & PstI
2 log DNA ladder
Result:
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
Sunday, July 14th
[Expand] Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct)
Investigator: Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P428 (mMCSII_t35S_npt-casette) and P477 (pAct).
Procedure:
Reaction batch for P428 (MCS-t35S-npt):
volume
reagent
15 µl
P428
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
XbaI (20 U/µl)
19 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P477 (pAct):
volume
reagent
15 µl
P477
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
2 µl
SpeI (10 U/µl)
18 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 4 h.
Preparative gelelectrophoresis was performed at 90 V for 2 h in an 1% agarose gel.
Lane
Digestion of P486 with XbaI & AgeI
2 log DNA ladder
Digestion of P488 with XbaI & PstI
2 log DNA ladder
Results
as expected; intense band was cut out
as expected; lowest band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Ligation of F185+F186 (pAct + MCS_t35S_npt)
Investigator: Jeff
Procedure:
Ligation batch for F185+F186 (pAct + MCS_t35S_npt), vector:insert = 1:3:
volume
reagent
4 µl
F185 (5000 bp)
4 µl
F186 (1200 bp)
1 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=10 µl
TOTAL
Ligation was performed at 22 °C for 1 h
Negative controls were also prepared with water instead of insert.
[Expand] Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1)
Investigator: Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES), P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES), P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA) and P510 (SERK-SigP_PP1).
Procedure:
Reaction batch for P430 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES):
volume
reagent
20 µl
P430
4 µl
CutSmart Buffer (10x)
2 µl
SpeI (20 U/µl)
1 µl
PstI-HF (20 U/µl)
13 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P431 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES):
volume
reagent
20 µl
P431
4 µl
CutSmart Buffer (10x)
2 µl
SpeI (10 U/µl)
1 µl
PstI-HF (20 U/µl)
13 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P509 ((GGGGS)x5-TEV-site-linker_NLS_NucA):
volume
reagent
20 µl
P509
4 µl
CutSmart Buffer (10x)
2 µl
NgoMIV (10 U/µl)
1 µl
PstI-HF (20 U/µl)
13 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P510 (SERK-SigP_PP1)):
volume
reagent
20 µl
P510
4 µl
CutSmart Buffer (10x)
1 µl
AgeI-HF (20 U/µl)
1 µl
PstI-HF (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P430 with XbaI & AgeI
2 log DNA ladder
Digestion of P431 with XbaI & PstI
Digestion of P509 with NgoMIV & SpeI
Results
as expected
as expected
as expected; lower band was cut out
Lane
2 log DNA ladder
Digestion of P510 with AgeI & PstI
Results
as expected
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
Aim of the experiment: Ligation of F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1).
Procedure:
Ligation batch for F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), vector:insert = 1:3:
volume
reagent
1.99 µl
F122 (50.3 ng/µl, 2369 bp)
0.98 µl
F189 (79.9 ng/µl, 618 bp)
14.03 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), vector:insert = 1:3:
volume
reagent
0.45 µl
F190 (224.7 ng/µl, 3155 bp)
5.32 µl
F135 (17.8 ng/µl, 996 bp)
11.23 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 16 °C overnight.
Negative controls were also prepared with water instead of insert.
Week 13
Monday, July 15th
[Expand] Transformation of the genes to be expressend in moss (retransformation) and F122+F189 (SERK-SigP_SERK-TMD + (GGGGS)x5-TEV-site-linker_NLS_NucA), F190+F135 (SERK-SigP_PP1 + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1)
Investigator: Jeff
Procedure for the retransformations:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
0.5 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
50 µl of this cell suspension was plated on chlorampenicol plates.
Tuesday, July 16th
[Expand] Preparative digestion and gelelectrophoresis of all effectors
Investigator: Jeff
Procedure:
Mastermix:
volume
reagent
224
ddH2O
64 µl
CutSmart Buffer (10x)
16 µl
EcoRI-HF (20 U/µl)
16 µl
PstI-HF (20 U/µl)
20 µl of DNA were mixed with 20 µl of Mastermix and were incubated at 37 °C for 4 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
2 log DNA ladder
Digestion of P20 (GFP) with EcoRI & PstI
2 log DNA ladder
Digestion of P196 (XylE) with EcoRI & PstI
2 log DNA ladder
Digestion of P245 (EreB) with EcoRI & PstI
Results
as expected
as expected
as expected
as expected
as expected
as expected
Lane
2 log DNA ladder
Digestion of P246 (nLuc) with EcoRI & PstI
2 log DNA ladder
Digestion of P330 (SERK-SigP_NLuc) with EcoRI & PstI
2 log DNA ladder
Digestion of P346 (Dehydrochlorinase) with EcoRI & PstI
Results
as expected
as expected
as expected
as expected
as expected
as expected
The digestion of P350 was separated for an additional hour because there might be contaminations of the vector that is smaller than the protein-coding BioBrick we want to express. The separated bands looked like that:
Lane
2 log DNA ladder
Digestion of P350 (EreB-Receptor) with EcoRI & PstI
2 log DNA ladder
Digestion of P354 (nLuc-Receptor) with EcoRI & PstI
2 log DNA ladder
Digestion of P402 (IgK-nLuc) with EcoRI & PstI
Results
as expected
as expected
as expected
as expected
as expected
as expected
Lane
2 log DNA ladder
Digestion of P476 (SpyCatcher-Receptor_IRES_IgK_SpyTag-nLuc) with EcoRI & PstI
2 log DNA ladder
Digestion of P496 (SpyTag-Receptor_IRES_nLuc_SpyCatcher with EcoRI & PstI
2 log DNA ladder
Digestion of P498 (SpyCatcher-Receptor_IRES_IgK_nLuc-SpyTAG) with EcoRI & PstI
Results
as expected
as expected
as expected
as expected
as expected
as expected
Lane
2 log DNA ladder
Digestion of P502 (IgK-GFP) with EcoRI & PstI
2 log DNA ladder
Digestion of P512 (SpyTAG-Receptor-IRES_SpyCatcher-nLuc) with EcoRI & PstI
2 log DNA ladder
Digestion of P516 (FluA-Recetpro) with EcoRI & PstI
Results
as expected
as expected
as expected
as expected
as expected
as expected
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
[Expand] Miniprep of P520 to P525: P520 (Bba_KK509000,Cauliflower Mosaic Virus 35S promoter) P521-525 (pAct(with_del)-MCS-T35S-npt
Investigator: Jeff
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P520 to P525.
[Expand] Analytical digestion and gelelectrophoresis of P520 - P525
Investigator: Jeff
Procedure:
7x Mastermix (EcoRI-HF & PstI-HF) for analytical digestion:
volume
reagent
7 µl
Cut Smart buffer
1.75 µl
EcoRI-HF (20 U/µl)
1.75 µl
PstI-HF (20 U/µl)
38.5 µl
ddH2O
=49 µl
TOTAL
7 µl master mix and 3 µl plasmid DNA were mixed.
Analytical digestion was performed at 37 °C for 1.5bsp;h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2 log DNA ladder
P428, dsted with EcoRI & PstI
P522, digested with EcoRI & PstI
P522, digested with EcoRI & PstI
P523, digested with EcoRI & PstI
P524, digested with EcoRI & PstI
P525, digested with EcoRI & PstI
Result:
control
no insert ;(
no insert ;(
no insert ;(
no insert ;(
no insert ;(
no insert ;(
Wednesday, July 17th
Thursday, July 18th
[Expand] Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P537 – P540 and analytical gelelectrophoresis of F206 and F207.
Procedure:
volume
reagent
2.5 µl
Plasmid DNA (P537 – P540)
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2 log DNA ladder
Digestion of P537 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI
Digestion of P538 F122+F189 (SERK-SigP_SERK-TMD in pSB1C3 + (GGGGS)x5-TEV-site-linker_NLS_NucA) with EcoRI & PstI
Digestion of P539 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI
Digestion of P540 F190+F135 (SERK-SigP_PP1 in pSB1C3 only + Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1) with EcoRI & PstI
F206
F207
Result:
as expected (916 bp)
as expected (916 bp)
as expected (2080 bp)
as expected (2080 bp)
as expected (3484 bp)
as expected (3484 bp)
[Expand] PCR to insert 60 missing base-pairs into pActin (new method)
Investigator: Jeff
Aim of the experiment: PCR to insert 60 missing base-pairs into pActin (new method)
Procedure:
Reaction batch for PCR with Q5 Mastermixes for P477 (pActin):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl
10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl
Plasmid DNA (P477) - 1:1000 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O114 (pAct_ins_fw)
2.5 µl
10 µM Reverse Primer O115 (pAct_ins_rv)
1 µl
Plasmid DNA (P477) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme:
Cycling parameters:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
72 °C
30 s
72 °C
2 min
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting product was labeled as F206 for the 1:1000 batch and F207 for the 1:100 batch.
Friday, July 19th
Saturday, July 20th
Sunday, July 21st
Week 14
Monday, July 22nd
[Expand]Arrival of the correct pActin 5 from Physcomitrella patens from Reski lab
This plasmid was named p541
Tuesday, July 23rd
Wednesday, July 24th
[Expand] PCR of P541 (PppActin5)
Investigator: Jeff
Aim of the experiment: PCR of P541 (PppActin5).
Procedure:
Reaction batch for PCR with Q5 Mastermix for P541 (PppActin5):
volume
reagent
25 µl
2x Q5 Master Mix
1 µl
dNTP mix (normally not necessary!)
2.5 µl
10 µM Forward Primer O116 (PppAct_f)
2.5 µl
10 µM Reverse Primer O117 (PppAct_r)
1 µl
Plasmid DNA (P541) - 1:1000 dilution (desired c between 1 pg and 1 ng)
18 µL
ddH2O Water
=50 µL
TOTAL
The content has been mixed with a pipette
The PCR program was performed after following scheme:
Cycling parameters:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
53 °C
30 s
72 °C
30 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting product was labeled as F208.
[Expand] Analytical gelelectrophoresis of F208
Investigator: Jeff
Aim of the experiment: Analytical gelelectrophoresis of F208.
Procedure:
5 µl of the PCR product F208 (=PCR of P541 with O116/O117) products were mixed with 2 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 60 min on a 1% agarose gel.
Lane:
2 log DNA ladder
F208
Result:
as expected
[Expand] Preparative digestion of P208 (PCR of PppActin5)
Investigator: Jeff
Procedure:
Reaction batch for F208 (PCR of PppActin5):
volume
reagent
25 µl
F208
5 µl
CutSmart Buffer (10x)
1 µl
EcoRI-HF
1 µl
SpeI-HF
18 µl
ddH2O
=45 µl
TOTAL
Reaction batch was incubated at 37 °C for 2 h.
After digestion, the product was purified with QIAquick PCR Purification Kit, Qiagen.
[Expand] Ligation and transformation of F209 + F41 (PppActin5 in pSB1C3)
Investigator: Jeff
Procedure:
Ligation batch for F209 (PppActin5), vector:insert = 1:3:
volume
reagent
7 µl
F209 (36.9 ng/µl, ??? bp)
1 µl
F189 (89.9 ng/µl, ??? bp)
1 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=10 µl
TOTAL
Ligation was performed at RT for 1 hour.
Negative controls were also prepared with water instead of insert.
The ligation products were transformed into E. coli XL-1 Blue.
Thursday, July 25th
[Expand] Picking of E. coli XL1 blue transformed with ligation product of F208 and F41 (PppActin5 in pSB1C3)
Investigator: Jeff
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
6 colonies were picked for ligation of F208 and F41 (PppActin5 in pSB1C3).
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Friday, July 26th
[Expand] Miniprep of P542 to P547 (pSB1C2_PppActin5)
Investigator: Jeff
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P542 to P547.
[Expand] Analytical digestion and gelelectrophoresis of P542 – P547
Investigator: Jeff
Procedure:
volume
reagent
3 µl
Plasmid DNA (P542 – P547)
1 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
5.5 µl
ddH2O
=10 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2 log DNA ladder
Digestion of P542 (PppActin5) with EcoRI & PstI
Digestion of P543 (PppActin5) with EcoRI & PstI
Digestion of P544 (PppActin5) with EcoRI & PstI
Digestion of P545 (PppActin5) with EcoRI & PstI
Digestion of P546 (PppActin5) with EcoRI & PstI
Digestion of P547 (PppActin5) with EcoRI & PstI
Result:
result unclear because insert and backbone have the same length!
result unclear because insert and backbone have the same length!
result unclear because insert and backbone have the same length!
result unclear because insert and backbone have the same length!
result unclear because insert and backbone have the same length!
result unclear because insert and backbone have the same length!
Unfortunately the correctness of the clones could not be seen in this experiment.
Thus a second digestion was performed:
volume
reagent
3 µl
Plasmid DNA (P542 – P547)
1 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
0.25 µl
NcoI (form Fermentas
5.25 µl
ddH2O
=10 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2 log DNA ladder
Digestion of P542 (PppActin5) with EcoRI & PstI
Digestion of P543 (PppActin5) with EcoRI & PstI
Digestion of P544 (PppActin5) with EcoRI & PstI
Digestion of P545 (PppActin5) with EcoRI & PstI
Digestion of P546 (PppActin5) with EcoRI & PstI
Digestion of P547 (PppActin5) with EcoRI & PstI
Result:
Clone correct: backbone and PppActin5
Clone wrong: only backbone, no insert
Clone correct: backbone and PppActin5
Clone correct: backbone and PppActin5
Clone wrong: only backbone, no insert
Clone correct: backbone and PppActin5
[Expand] Sequencing of P544 and P545
Investigator: Ingmar
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1
Barcode2
P544
FR02233095
FR02233087
P545
FR02233093
FR02233094
Saturday, July 27th
Sunday, July 28st
Week 15
Monday, July 29th
[Expand] QuikChange of P544 (PppActin5)
Investigator: Jeff
Procedure:
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P544 template (1:100)
1.0 µl
1:10 dilution of O118 (=10 µM)
1.0 µl
1:10 dilution of O119 (=10 µM)
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl
ddH2O
=25 µl
TOTAL
volume
reagent
2.5 µl
10x Pfu Ultra II buffer
4 µl
Plasmid P544 template (1:100)
1.0 µl
1:10 dilution of O120 (=10 µM)
1.0 µl
1:10 dilution of O121 (=10 µM)
1 µl
dNTP mix 50 mM
0.5 µl
Pfu Ultra II DNA polymerase (2.5 U/µl)
15 µl
ddH2O
=25 µl
TOTAL
PCR cycling parameters with 55 °C annealing temperature:
Cycles
Temperature
Time
1
95 °C
30 sec
10
95°C
30 sec
55°C
1 min
72°C
5 min
1
4°C
HOLD
Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the pooled reaction batches and incubate for 1 h at 37 °C.
Tuesday, July 30th
Wednesday, July 31st
[Expand] Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P537 (SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
Reaction batch for P537 (SERK-SigP_SERK-TMD_(GGGGS)-TEV-site-linker_NLS_NucA):
volume
reagent
20 µl
P537
4 µl
CutSmart Buffer (10x)
2 µl
XbaI (20 U/µl)
2 µl
PstI-HF (20 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
2 log DNA ladder
Digestion of P537 with XbaI & PstI
Results
as expected; lower band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
Resulting fragment was named as F210.
[Expand] Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Ligation of F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
Ligation batch for F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:
volume
reagent
0.53 µl
F187 (189.9 ng/µl, 7346 bp)
0.68 µl
F210 (55.3 ng/µl, 916 bp)
15.79 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA, vector:insert = 1:3:
volume
reagent
0.73 µl
F188 (136.7 ng/µl, 7346 bp)
0.68 µl
F210 (55.3 ng/µl, 916 bp)
15.59 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 16 °C overnight.
Negative controls were also prepared with water instead of insert.
Thursday, August 1st
[Expand] Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-Blue with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
Friday, August 2nd
[Expand] Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Picking of E. coli XL1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
Picked colonies were transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contains 4 mL of LB-medium + 4 µL chloramphenicol (1000x).
2 colonies were picked for BBa_K864100, BBa_E0020, F187+F210 and F188+F210 each.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated at 180 rpm overnight.
Saturday, August 3rd
[Expand] Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with BBa_K864100 (SYFP2), BBa_E0020 (eCFP) and ligation products F187+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and F188+F210 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES in pSB1C3 + SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P558 to P565.
[Expand] Analytical digestion and gelelectrophoresis of P558 – P565
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P558 – P565.
Procedure:
Mastermix for analytical digestion with EcoRI and PstI
volume
reagent
20 µl
Cut Smart buffer
2.5 µl
EcoRI-HF
2.5 µl
PstI-HF
150 µl
ddH2O
=175 µl
TOTAL
17.5 µl of the mastermix were mixed together with 2.5 µl of Plasmid DNA (P558 – P565)
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
Digestion of P558 (BBa_K864100)
Digestion of P559 (BBa_K864100)
2 log DNA ladder
Digestion of P560 (BBa_E0020)
Digestion of P561 (BBa_E0020)
Result:
as expected (723 bp)
as expected (723 bp)
as expected (723 bp)
as expected (723 bp)
Lane:
Digestion of P562 (F187+F210)
Digestion of P563 (F187+F210)
2 log DNA ladder
Digestion of P564 (F188+F210)
Digestion of P565 (F188+F210)
Result:
as expected (6191 bp)
as expected (6191 bp)
not as expected; corrupt (6191 bp)
as expected (6191 bp)
Sunday, August 4th
Week 16
Monday, August 5th
[Expand] Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA)
Investigator: Jeff
Aim of the experiment: Preparative digestion and gelelectrophoresis of P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P558 (SYFP2), P560 (eCFP), P562 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA) and P565 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_SERK-SigP_SERK-TMD_(GGGGS)x5-TEV-site-linker_NLS_NucA).
Procedure:
Reaction batch for P540:
volume
reagent
20 µl
P540
4 µl
CutSmart Buffer (10x)
2 µl
EcoRI-HF (20 U/µl)
2 µl
PstI-HF (20 U/µl)
4 µl
XhoI (10 U/µl)
8 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P558, P560, P562, P565:
volume
reagent
20 µl
P558, P560, P562, P565
4 µl
CutSmart Buffer (10x)
2 µl
EcoRI-HF (20 U/µl)
2 µl
PstI-HF (20 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P540 with EcoRI & PstI & XhoI
2 log DNA ladder
Digestion of P558 with EcoRI & PstI
Digestion of P560 with EcoRI & PstI
Results
as expected; upper band was cut out
as expected; lower band was cut out
as expected; lower band was cut out
Lane
Digestion of P562 with EcoRI & PstI
2 log DNA ladder
Digestion of P565 with EcoRI & PstI
Results
as expected; upper band was cut out
as expected; upper band was cut out
DNA bands were extracted with QIAquick Gel Extraction Kit, QIAGEN.
Resulting fragment was named as F214 – F218.
[Expand] PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes
Investigator: Jeff
Aim of the experiment: PCR of P558 (SYFP2) and P560 (eCFP) to introduce RFC25 pre- and suffixes.
Procedure:
Reaction batch for PCR with Q5 Mastermixes for P558 (SYFP2):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O122 (SYFP2_fw)
2.5 µl
10 µM Reverse Primer O123 (SYFP2_rv)
1 µl
Plasmid DNA (P558) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
Reaction batch for PCR with Q5 Mastermixes for P560 (eCFP):
volume
reagent
25 µl
2x Q5 Master Mix
2.5 µl
10 µM Forward Primer O124 (eCFP_fw)
2.5 µl
10 µM Reverse Primer O125 (eCFP_rv)
1 µl
Plasmid DNA (P560) - 1:100 dilution (desired c between 1 pg and 1 ng)
19 µL
ddH2O Water
=50 µL
TOTAL
The content has been created on ice and mixed with a pipette
The gradient PCR program was performed after following scheme with following conditions (Tm=67 °C; ΔG=2 °C; P558 in row 1(=65.0 °C); P560 in row 12 (=69.2 °C)):
Cycling parameters:
Initial denaturation
98 °C
30 s
30 cycles
98 °C
10 s
Tm=67 °C; ΔG=2 °C
30 s
72 °C
22 s
Final extension
72 °C
2 min
Hold
4 °C
infinite
After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.
The resulting product was labeled as F212 for the the PCR products of P558 and F213 for the PCR product of P560.
[Expand] Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)
Investigator: Jeff
Aim of the experiment: Analytical gelelectrophoresis of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).
Procedure:
4.5 µl of the PCR product F212 and F213 products were mixed with 0.5 µl of DNA loading buffer (10x).
Analytical gelelectrophoresis was performed at 90 V for 60 min on an 1% agarose gel.
Lane:
F212
2 log DNA ladder
F213
Result:
as expected
as expected
Tuesday, August 6th
[Expand] Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv)
Investigator: Jeff
Aim of the experiment: Oligohybridization of O128(G-TEV-site-G_fw) and O129 (G-TEV-site-G_rv).
Procedure:
25 µL of O128 (100 µM) and 25 µL of O129 (100 µM) were pooled together.
Heating up to 95 °C for 5 min.
Cooling at RT in a styropor box overnight.
[Expand] Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25)
Investigator: Jeff
Aim of the experiment: Preparative digestion of F212 (SYFP2 RFC25) and F213 (eCFP RFC25).
Procedure:
Reaction batch for F212:
volume
reagent
25 µl
F212
5 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
AgeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Reaction batch for F213:
volume
reagent
25 µl
F213
5 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
AgeI-HF (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Reaction batches were incubated at 37 °C for 2 h.
Digested products were purified with QIAquick PCR Purification Kit, QIAGEN.
[Expand] Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP)
Investigator: Jeff
Aim of the experiment: Ligation of F123+F219 (pSB1C3 + SYFP2) and F123+F220 (eCFP).
Procedure:
Ligation batch for F123+F219 (pSB1C3 + SYFP2), vector:insert = 1:3:
volume
reagent
1 µl
F123 (49.3 ng/µl, 2086 bp)
0.4 µl
F219 (114.9 ng/µl, 723 bp)
15.6 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F123+F220 (pSB1C3 + eCFP), vector:insert = 1:3:
volume
reagent
1 µl
F123 (49.3 ng/µl, 2086 bp)
1.2 µl
F220 (41.7 ng/µl, 723 bp)
14.8 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at 16 °C overnight.
[Expand] Analytical digestion and gelelectrophoresis of further clones
Investigator: Jeff
Procedure:
The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.
Analytical digestion was performed at 37 °C for 2 h.
After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
Result:
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
[Expand] Preparative digestion of P428 (mMCS-T35S-npt) and P554 (PppAct5-QCII)
Investigator: Jeff
Procedure:
Reaction batch for P428:
volume
reagent
20 µl
P428
5 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
XbaI (20 U/µl)
23 µl
ddH2O
=50 µl
TOTAL
Reaction batch for P554:
volume
reagent
20 µl
F213
5 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
2 µl
SpeI (10 U/µl)
2 µl
NcoI (10 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Reaction batches were incubated at 37 °C for 2 h.
Lane
2 log DNA ladder
Digestion of P428 with EcoRI & XbaI
Digestion of P554 with EcoRI & SpeI
Results
as expected; highest band was cut out
as expected; highest band was cut out
The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.
[Expand] Ligation of F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt)
Investigator: Jeff
Procedure:
Ligation batch for F221+F222 (PppAct5 + pSB1C3-mMCS-T35S-npt), vector:insert = 1:3:
volume
reagent
5 µl
F221 (42 ng/µl, 3700 bp)
11 µl
F222 (37 ng/µl, 2000 bp)
2 µl
T4 ligase buffer (10x)
2 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at room temperature overnight.
[Expand] Transformation of E. coli XL1-Blue with ligation F221+F222
Investigator: Jeff
Procedure:
After 1 h of ligation 10 µl were used for transformation.
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
Wednesday, August 7th
[Expand] Transformation of E. coli XL1-Blue with ligation F221+F222
Investigator: Jeff
Procedure:
After 1 h of ligation 10 µl were used for transformation.
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 100 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed after 1 h
Investigator: Jeff
Procedure:
PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
12 colonies were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight
Thursday, August 8th
[Expand] Analytical digestion and gelelectrophoresis of further clones
Investigator: Jeff
Procedure:
The plasmids were cut with 0.2 µl of EcoRI and SpeI in a volume of 10 µl.
Analytical digestion was performed at 37 °C for 2 h.
After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
Result:
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
All these clones were discarded.
Additionally the fragments for the ligation were controlled.
Lane:
2log DNA ladder
F185
F211
F221
F222
Result:
correct
correct
correct
correct
[Expand] Picking of of E. coli XL1 blue with PppAct5-mMCS-T35S-npt from the ligation performed over night
Investigator: Jeff
Procedure:
PppAct5-mMCS-T35S-npt: Colonies were picked from chloramphenicol plates.
Picked pipette tips was transferred into cell-culture tubes with air-permeable, sterile cover. Each tube contain 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
40 colonies were picked.
These tubes were transferred in a cell culture shaker at 37 °C and were incubated overnight
Friday, August 9th
[Expand] Miniprep of E. coli Xl1-Blue transformed with ligation of PppAct5 into mMCS-T35S-npt
Investigator: Jeff
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P574 to P613.
[Expand] Analytical digestion and gelelectrophoresis of P574 – P613
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P574 – P613.
Procedure:
Mastermix for analytical digestion with EcoRI
volume
reagent
45 µl
Cut Smart buffer
8 µl
EcoRI-HF
400 µl
ddH2O
=453 µl
TOTAL
9 µl of the mastermix were mixed together with 1 µl of Plasmid DNA
Analytical digestion was performed at 37 °C for 2 h.
After incubation 3 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
Result:
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
good clone => only faint additional band
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
Result:
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
best clone => no additional band (best clone)
wrong result
wrong result
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
Result:
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
wrong result
correct size, but additional bands
correct size, but additional bands
Lane:
2log DNA ladder
clone 1
clone 2
clone 3
clone 4
clone 5
clone 6
clone 7
clone 8
clone 9
clone 10
Result:
wrong result
wrong result
correct size, but additional bands
wrong result
wrong result
correct size, but additional bands
wrong result
wrong result
wrong result
wrong result
Saturday, August 10th
[Expand] Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25)
Investigator: Jeff
Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation products F123+F219 (SYFP RFC25) and F123+F220 (eCFP RFC25).
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P614 to P617.
[Expand] Analytical digestion and gelelectrophoresis of P614 – P617
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P614 – P617.
Procedure:
Mastermix for analytical digestion with EcoRI and PstI
volume
reagent
2.5 µl
Plasmid DNA (P614 – P617)
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25nbsp;µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 120 V for 30 min.
Lane:
2 log DNA ladder
Digestion of P614 (SYFP2 RFC25)
Digestion of P615 (SYFP2 RFC25)
Digestion of P616 (eCFP RFC25)
Digestion of P617 (eCFP RFC25)
Result:
as expected (723 bp)
as expected (723 bp)
as expected (723 bp)
as expected (723 bp)
Sunday, August 11th
[Expand] Preparative digestion of P615 (SYFP2 RFC25)
Investigator: Jeff
Aim of the experiment: Preparative digestion of P615 (SYFP2 RFC25)
Procedure:
[Expand] Preparative digestion and gelelectrophoresis of P617 (eCFP RFC25)
Investigator: Jeff
Aim of the experiment: Preparative digestion of P617 (eCFP RFC25)
Procedure:
[Expand] Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2)
Investigator: Jeff
Aim of the experiment: Ligation of F224+F223 (creation of Gly-TEV-site-Gly_SYFP2).
Procedure:
Ligation batch for F224+F223 (SYFP2 + Gly-TEV-site-Gly), vector:insert = 1:3:
Aim of the experiment: Sequencing of P509, P512, P516, P537, P540, P562, P565, P583, P591
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode1 (fw)
Barcode2 (rev)
P509
FR02233103
P632
FR02233102
FR02233101
P633
FR02233100
FR02233099
P537
FR02233098
FR02233097
P540
FR02233096
FR02233104
P562
FR02233105
FR02233106
P565
FR02233107
FR02233108
P583
FR02233109
FR02233110 (Primer O131)
P591
FR02233111
FR02233088 (Primer O131)
[Expand] Transformation of E. coli XL1-Blue with P337,P540, P583 and P591
Investigator: Johanna
Aim of the experiment: Retransformation of E. coli XL1-Blue with P337,P540, P583 and P591
Procedure:
1,5 µl of dd H2O were added to the empty tubes
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Transformation of E. coli XL1-Blue with ligation product F224+F223 (SYFP2 + G-TEV-site-G) and negative control of F224
Investigator: Louise
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F224+F223 (SYFP2 + G-TEV-site-G) and negative control of F224
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
Tuesday, August 13th
[Expand] Gelextraction and Ligation of digested P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt)
Investigator: Louise
Aim of the experiment: Gelextraction and selfligation to verify correct cut of digested P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt)
Procedure:
After preparative digestion of P583 and P591 with SpfI and MfeI the gel extraction was performed after manufacturer`s protocol (QIAquick Gel Extraction Kit, QIAGEN)
the extracted fragments were labelled F226 (digested P583) and F227 (digested P591)
ligation was performed without extra inserts with just 100 ng of cut plasmid to test if it was cut as expected
Ligation batch for self-ligation of F226
volume
reagent
0.6 µl
F226 (164.1 ng/µl)
16.4 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for self-ligation of F227
volume
reagent
0.4 µl
F227 (243.4 ng/µl)
16.6 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
[Expand] Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_J45014
Investigator: Louise
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product of self-ligated F226 and F227 and with Biobrick BBa_245014
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Picking of Retrafos of P337, P540, P583 and P591 and of Ligation F224 + F223
Investigator: Jeff
Aim of the experiment: Picking of Retrafos of P337 (SERK-SigP_XylE), P540 (SERK-SigP_PP1_Strep-tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1), P583 (PppActin5-MCS-T35S-npt) and P591 (PppActin5-MCS-T35S-npt) and of Ligation F224 + F223 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)
Procedure:
Retrafos 1 clone each, Ligation 4 clones due to approx. 50 % positive neg. ctrl
4 ml LB medium w/ 4 µl chloramphenicol
Incubation overnight at 37 °C in the 180 rpm
Wednesday, August 14th
[Expand] Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)
Investigator: Louise
Aim of the experiment: Picking of of E. coli XL1 blue transformed with BBa_J45014 (banana odour)
Procedure:
BBa_J45014 (banana odour): 4 Colonies were picked from chloramphenicol plates.
Each colony was transferred into a tube containing 4 mL of LB-medium + 4 µL chloramphenicol(1000x).
These tubes were transferred into a cell culture shaker at 37 °C and were incubated overnight
[Expand] Analytical digestion and gelelectrophoresis of P640-P643 (G-TEV-site-G_SYFP2) and P615 with EcoRI and PstI and P637, P639 with EcoRI and MfeI (old and new)
Investigator: Louise, Johanna
Aim of the experiment: Analytical digestion and gelelectrophoresis of P640-P643 (G-TEV-site-G_SYFP2) and P615 (SYFP2, RFC25) with EcoRI and PstI and P637 (PppActin5-MCS-T35S-npt), P639 (PppActin5-MCS-T35S-npt) with EcoRI and MfeI (old and new)
Procedure:
batch for digestion of P640-P643
volume
reagent
8.5 µl
Plasmid DNA
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
9 µl
ddH2O
=20 µl
TOTAL
batch for digestion of P615
volume
reagent
2.5 µl
Plasmid DNA
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
batch for digestion of P637 and P639
volume
reagent
8.5 µl
Plasmid DNA
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
MfeI (old or new)
9 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
DNA ladder
Digestion of P615 (SYFP2 RFC25-reference)
Digestion of P637 (with old MfeI)
Digestion of P637 (with new MfeI)
Digestion of P639 (with old MfeI)
Digestion of P639 (with new MfeI)
Digestion of P640
Digestion of P641
Digestion of P642
Digestion of P643
Result:
as expected
corrupt MfeI
as expected
corrupt MfeI
as expected
as expected
as expected
as expected
as expected
[Expand] Sequencing of P614, P617, P637, P639 and P641
Investigator: Flo, Johanna
Aim of the experiment: Sequencing of P614, P617, P637, P639 and P641
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode
Primer
P614
FR02233112
03
P617
FR02233113
03
P641
FR02233114
03
P637
FR02233119
061
P639
FR02233115
061
[Expand] Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223
Investigator: Johanna
Aim of the experiment: Miniprep of Retrafos of P337, P583, P540, P591 and of ligation product F224+F223
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The new plasmids were numbered as followed:
Plasmid
Miniprep-Plasmid
P337
P636
P583
P637
P540
P638
P591
P639
F224+223 (version 1)
P640
F224+223 (version 2)
P641
F224+223 (version 3)
P642
F224+223 (version 4)
P643
[Expand] Miniprep of Retrafos of banana odour, P583 and P591
Investigator: Florian
Aim of the experiment: Miniprep of Retrafos of banana odour, P583 and P591.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The bands were extracted from the gel and termed F221 and F222. The concentrations were determined to 42 and 37 ng/µl.
Thursday, August 15th
[Expand] Preparative digestion of P414 and P640 + Preparative Gelelectrophoresis + Gelextraction
Investigator: Andi
Procedure:
Reaction batch for P414:
volume
reagent
20 µl
P414
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P640:
volume
reagent
20 µl
P640
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
2 µl
NgoMIV (20 U/µl)
13 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 1% agarose gel.
Lane
Digestion of P414 with EcoRI & PstI
1kB DNA ladder
Digestion of P640 with EcoRI & NgoMIV
Results
as expected; upper band was cut out
as expected; lower band was cut out
Gelextraction was performed after preparative Gelelectrophoresis
The resulting fragments were named F231 (Laccase) + F232
[Expand] Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218
Investigator: Louise, Florian
Aim of the experiment: Ligation of F230 with F191, F192, F193, F194, F195, F196, F197, F198, F199, F200, F201, F202, F203, F204, F205, F214, F217 and F218
Procedure:
Ligation batch for F230+F191 (plasmid with mini MCS + GFP cytoplasmatic), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
18.7 µl
F191 (2 ng/µl, 723 bp)
0.0 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F192 (plasmid with mini MCS + catecholdioxygenase cytoplasmatic), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
5.94 µl
F192 (8 ng/µl, 918 bp)
10.65 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F193 (plasmid with mini MCS + EreB cytoplasmatic), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
1.85 µl
F193 (35 ng/µl, 1254 bp)
14.74 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F194 (plasmid with mini MCS + Nanoluciferase cytoplasmatic), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
8.80 µl
F194 (3 ng/µl, 510 bp)
7.79 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F195 (plasmid with mini MCS + nanoluciferase secretory_SERK), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
3.99 µl
F195 (8 ng/µl, 616 bp)
12.60 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F196 (plasmid with mini MCS + gutathiontransferase cytoplasmatic), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
1.17 µl
F196 (28 ng/µl, 633 bp)
15.42 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F197 (plasmid with mini MCS + EreB membrane associated), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
11.12 µl
F197 (11 ng/µl, 2362 bp)
5.47 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F198 (plasmid with mini MCS + Nanoluc membrane associated), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
6.44 µl
F198 (13 ng/µl, 1618 bp)
10.15 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F199 (plasmid with mini MCS + nanoluc secretory_Ig Kappa), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
1.16 µl
F199 (26 ng/µl, 583 bp)
15.43 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F200 (plasmid with mini MCS + Nanoluc+IRES+Spytag_Nanoluc), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
3.00 µl
F200 (47 ng/µl, 2725 bp)
13.59 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F201 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spycatcher), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
3.81 µl
F201 (37 ng/µl, 2725 bp)
12.78 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F202 (plasmid with mini MCS + Nanoluc+IRES+Nanoluc_Spytag), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
2.66 µl
F202 (53 ng/µl, 2725 bp)
13.93 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F203 (plasmid with mini MCS + IgKappa_GFP secretory), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
1.09 µl
F203 (38 ng/µl, 800 bp)
15.50 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F204 (plasmid with mini MCS + Nanoluc+IRES+Spycatcher_Nanoluc), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
2.17 µl
F204 (65 ng/µl, 2725 bp)
14.42 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F205 (plasmid with mini MCS + FluA membrane associated), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
2.11 µl
F205 (40 ng/µl, 1630 bp)
14.48 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F214 (plasmid with mini MCS + PP1 membrane associated), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
0.95 µl
F214 (113 ng/µl, 2080 bp)
15.64 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
2.76 µl
F217 (116 ng/µl, 6191 bp)
13.83 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F217 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1
volume
reagent
1.15 µl
F230 (243.7 ng/µl, 5796 bp)
0.86 µl
F217 (116 ng/µl, 6191 bp)
14.99 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F218 (plasmid with mini MCS + PIF6 safety), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
2.50 µl
F218 (128 ng/µl, 6191 bp)
14.09 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F218 (plasmid with mini MCS + PIF3 safety), vector:insert = 3:1
volume
reagent
1.15 µl
F230 (243.7 ng/µl, 5796 bp)
0.78 µl
F218 (116 ng/µl, 6191 bp)
15.07 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at roomtemperature, 1 h
[Expand] Ligation of F230 with F231
Investigator: Louise, Christopher
Aim of the experiment: Ligation of F230 with F231
Procedure:
Ligation batch for F230+F231 (plasmid with mini MCS + Laccase membrane-associated), vector:insert = 1:3
volume
reagent
0.41 µl
F230 (243.7 ng/µl, 5796 bp)
14.33 µl
F231 (9.5 ng/µl, ~2630 bp)
2.26 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation was performed at room temperature, 1 h
[Expand] Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230
Investigator: Louise, Christopher
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F230+F191, F230+F192, F230+F193, F230+F194, F230+F195, F230+F196, F230+F197, F230+F198, F230+F199, F230+F200, F230+F201, F230+F202, F230+F203, F230+F204, F230+F205, F230+F214, F230+F217, F230+F218, F217+F230, F218+F230 and negative control of F230
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
Friday, August 16th
[Expand] Sequencing of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G)
Investigator: Rosario
Aim of the experiment: Sequencing of P640.
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode
Primer
P640
FR02233116
03
[Expand]Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV
Investigators: Louise, Jeff
Aim of the experiment: Preparative digestion of P640 (SYFP2 RFC25 in pSB1C3 + Oligohybridization of G-TEV-site-G) with EcoRI+NgoMIV
Procedure:
volume
reagent
33 µl
P640
4 µl
CutSmart Buffer (10x)
2 µl
NgoMIV (10 U/µl)
1 µl
EcoRI Hf (20 U/µl)
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 3 h.
Lane
2 log DNA ladder
Digestion of P640 with EcoRI & NgoMIV
Results
as expected; band was cut out
The band was extracted from the gel via QIAGEN Gel Extraction Kit and termed F233. The concentration was 31.1 ng/µl.
[Expand] Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3)
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F231(plasmid with mMCS+membrane-bound Laccase) and negative control of F230, Retrafo of P541 (PppActin5) and P449(stress inducible Promoter_RFP +t35S-NPT-pSB1C3).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
1,5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspension were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Ligation of F233+F225 (creation of eCFP_Gly-TEV-site-Gly_SYFP2)
Investigator: Jeff
Aim of the experiment: Creation of eCFP_Gly-TEV-site-Gly_SYFP2
Aim of the experiment: Midiprep of RFC25 compatible RFP generator in (pSB1C3)
Procedure:
Midiprep was performed after manufacturer's protocol (QIAprep Midiprep, QIAGEN)
[Expand] Digestion of BITTE VERVOLLSTÄNDIGEN
Investigator: Ingmar
Aim of the experiment:
Procedure:
Week 18
Monday, August 19th
[Expand] Analytical Gelelectrophoresis of digested Midi-Preps F243 - F260
Investigator: Ingmar
Aim of the experiment: Verification of linearisation of digest fragments F234-F260
Procedure:
Concentrations of digestions were measured (blanked against EB-buffer), see Inventory List
5 µl of DNA loading buffer (10x) mixed with 1 µl Midiprep-DNA, on 1% agarose gel.
90 V 1 h.
Lane
1kB DNA ladder
F234
F235
F236
F237
F238
F239
F240
F241
F242
F243
F244
Name
-
Nanoluc secretory
GFP cytoplasmatic
Nanoluc membrane bound
Nanoluc cytoplasmatic
Ere B cytoplasm
PIF6
PIF3 clone1
PIF3 clone2
GFP_IgKappa
PP1
C1
Results
Lane
1kB DNA ladder
F245
F246
F247
F248
F249
F250
F251
F252
Name
-
Safety_PIF3
PP1 mebrane bound
Safety_PIF6
Safety_PIF?
pos ctrl mit Ppp Actin5
Ere B cytoplasm
Nanoluc cytoplasm
XylE cytoplasm
Results
Lane
1kB DNA ladder
F253
F254
F255
F256
F257
F258
F259
F260
Name
-
Nanoluc secretory IgKappa
Nanoluc secretory SERK
GFP cytoplasm
GFP secretory IgKappa
FluA membrane bound
stress inducible promotor
GST
Nanoluc membrane bound
Results
-
[Expand] Preparation of the linearized DNA (F234-F260) for the Moss Transformation
Investigator: Ingmar
Aim of the experiment: Preparation of the linearized DNA (F234-F260) for the Moss Transfection
Procedure:
The DNA pellets were solved in 500 µl ddH2O
Linerization of the DNA with EcoRI: 56,6 µl CutSmart buffer and 10 µl EcoRI were added to the 500 µl DNA. Incubation over night at 37 °C.
For purification the DNA was again precipitated with pure isopropanol: Addition of 500 µl isopropanol, mixing by inverting, centrifugation at 16,200 g for 40 min; supernatant was discarded
Wash pellet with 200 µl 70 % ethanol, centrifugation at 16,200 g for 40 min, supernatant discarded. The pellet was air dried at 50 °C for 8 min.
The DNA pellets were dissolved in the respectively calculated volumes of 0,1 M Ca(NO3)2 soultion to obtain final concentrations of 250 ng/µl.
fragment
name
concentration
concentration w/ loss
volume 0,1 M Ca(NO3)2
F234
Nanoluc SERK-SigP
440,8
396,72
872,78
F235
GFP cytoplasm
241,8
217,62
478,76
F236
Nanoluc membrane
339,9
305,91
673,00
F237
Nanoluc cytoplasm
219,1
197,19
433,82
F238
EreB cytoplasm
355,9
320,31
704,68
F239
PIF6
669,9
602,91
1326,40
F240
PIF3 (1)
438,1
394,29
867,438
F241
PIF3 (2)
642,9
578,61
1272,942
F242
GFP IgKappa
339,4
305,46
672,012
F243
PP1 receptor
333,5
300,15
660,33
F244
Catecholdioxygenase cytoplasm
342,4
308,16
677,952
Tuesday, August 20th
[Expand] Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261
Investigator: Andi, Rosario, Johanna
Aim of the experiment: Preparative digestion + Gelelectrophoreses + Gelextraction of P643/F261.
Procedure:
Reaction batch for P643:
volume
reagent
20 µl
P643
5 µl
CutSmart Buffer (10x)
2 µl
EcoRI (20 U/µl)
2 µl
NgoMIV (20 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Result of the Gelelectrophoresis:
Reaction batch was incubated at 37 °C for 2 h.
Digested product was purified with QIAquick PCR Purification Kit, QIAGEN.
Gelelectrophoresis was performed for 1 h and 90V.
Gelextraction was performed after manufacturers protocol and the resulting Fragment was called F261.
[Expand] Sequencing of P643 (SYFP2+ G-TEV-site-G)
Investigator: Andi
Aim of the experiment: Sequencing of P643 (SYFP2+ G-TEV-site-G)
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The different genes we sequenced received the following barcodes:
Label
Barcode
Primer
P643
FR0223317
03
[Expand]Ligation of F261+F225 (G-TEV-site-G_SYFP2 + eCFP)
Investigator: Rosario, Andi, Johanna
Aim of the experiment: Ligation of F261+F225 (G-TEV-site-G_SYFP2 + eCFP)
Procedure:
Ligation batch for F261+F225, vector:insert = 1:3
volume
reagent
1,5 µl
F225
1,2 µl
F261
14,3 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. w/ water instead of insert
Ligation was performed at room temperature, 1 h
[Expand] Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261 and Retrafo of P643.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA (Ligation) and 1 µl of DNA (Retrafo) were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Transformation of Physcomitrella patens with F234-F260
Investigator: Ingmar, Jeff
Aim of the experiment: Transformation of Physcomitrella patens with F234-F260
Procedure:
According to the Reski protocol [http://www.plant-biotech.net/]
Prepare 4 % driselase solution in 0.5 M mannitol, vortex, keep on rotating platform for 45 min (protected from light) and centrifuge at 3500 rpm, 10 min; sterile filter
Extraction of moss from bioreactor culture by filtering through protoplast sieve (mesh size 100 µm)
Addition of 12 ml 0.5 M mannitol solution and 4 ml of driselase stock solution (final concentration: 1 % w/v) and Incubation for 2 h at room temperature on rotating platform (protected from light)
Gently filter moss material through protoplast sieve (mesh size 100 µm), then filtrate through 50 µm sieve
Centrifugation at 500 rpm for 10 min with slow acceleration rates in a glass tube and discard supernatant; wash protoplasts with mannitol solution, repeat.
Resuspend pellet in total 10 ml mannitol
Determine concentration of protoplasts/ ml
100 µl of 250 ng/µl DNA solution with 350 µl PEG 4000 solution (8 g PEG4000 ad 20 g 3M medium, sterile filtered), incubate 30 min while gently mixing every 5 min
Dilution with 3M medium every 5 min: 1 ml, then 2 ml, then 3, then 4
Centrifugation at 500 rpm for 10 min, discard supernatant, resuspend in 4 ml regeneration medium and divide into 2 6wells
Incubation in the dark over night, then light/dark regime of 16/8 h
Wednesday, August 21st
[Expand] Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643
Investigator: Rosario
Aim of the experiment: Picking of of E. coli XL1 blue transformed with ligation product F225+F261 and Retrafo of P643.
Procedure:
F225+F261: 3 Colonies were picked from chloramphenicol plates.
P643: 1 Colony was picked from a chloramphenicol plate.
Each colony was transferred into a tube containing a relation of 1000:1 of LB-medium : antibiotic
These tubes were transferred into a cell culture shaker at 37 °C and were incubated 12 h
[Expand] Transformation of E. coli XL1-Blue with ligation product F225+F261
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F225+F261.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
Thursday, August 22nd
[Expand] Miniprep of E. coli Xl1-Blue transformed with ligation product F261 + F225 and Retrafo of P643
Investigator: Florian
Aim of the experiment: Miniprep of E. coli Xl1-Blue transformed with ligation product F261 + F225 and Retrafo of P643.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P714 to P716.
[Expand] Analytical digestion and gelelectrophoresis of P714 & P715
Investigator: Florian
Aim of the experiment: Analytical digestion and gelelectrophoresis of P714 & P715.
Procedure:
Batch for digestion with EcoRI & PstI
volume
reagent
2.5 µl
Plasmid
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
P714
P715
Result:
as expected
as expected
[Expand] ReTrafo of E. coli XL1-Blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Investigator: Rosario
Aim of the experiment: ReTrafo of E. coli XL1-Blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA (Ligation) and 1 µl of DNA (Retrafo) were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand]Preparative digestion of P625, P414 with EcoRI, PstI and MlyI, P628, P629, P630, P632 with EcoRI, PstI and SspI + Preparative Gelelectrophoresis + Gelextraction
Investigator: Johanna, Louise
Procedure:
Reaction batch for P625, P414:
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
PstI (20 U/µl)
2 µl
MlyI (20 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P628,P629,P630, P632:
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
PstI (20 U/µl)
2 µl
SspI (20 U/µl)
12 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0.5% agarose gel.
Gelextraction was performed after preparative Gelelectrophoresis
The resulting fragments were named F262 (digestion of P625), F264 (digestion of P628), F263 (digestion of P629, F265 (digestion of P630), F266 (digestion of P632) and F267 (digestion of P414).
[Expand] Ligation of F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267
Investigator: Flo, Rosario, Jeff
Aim of the experiment: Ligation of F230+F262 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F263 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyCatcher in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F264 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyTag_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F265 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F266 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F267 (SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3)
Procedure:
Ligation batch for F230+F262, vector:insert = 1:3
volume
reagent
0.4 µl
F230
15.96 µl
F268
0.7 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F263
volume
reagent
0.4 µl
F230
16.6 µl
F263
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F264, vector:insert = 1:3
volume
reagent
0.4 µl
F230
5.9 µl
F264
10.7 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F265, vector:insert = 1:3
volume
reagent
0.4 µl
F230
3.6 µl
F265
13 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F266, vector:insert = 1:3
volume
reagent
0.4 µl
F230
2.5 µl
F266
14.1 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F267, vector:insert = 1:3
volume
reagent
0.4 µl
F230
9.8 µl
F267
6.8 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. water instead of insert.
Ligation was performed at room temperature for 1 h.
Friday, August 23rd
[Expand]Preparative digestion of P715 with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction
Investigator: Louise, Flo
Procedure:
Reaction batch for P715:
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% agarose gel.
The lower band was cut out
Gelextraction was performed after preparative Gelelectrophoresis
[Expand] Transformation of E. coli XL1-Blue with P6 (pGAL in pTUM 100)
Investigator: Louise
Aim of the experiment: Transformation of E. coli XL1-Blue with P6 (pGAL in pTUM 100).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new ampicillin plates.
[Expand] Sequencing of P715 (sYFP_G_TEV-site_G_cYFP)
Investigator: Louise
Aim of the experiment: Sequencing of P715 (sYFP_G_TEV-site_G_cYFP)
Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).
The gene we sequenced received the following barcode:
Label
Barcode
Primer
P715
FR02233118
03
[Expand]Ligation of F187+F268 and F188+F268
Investigator: Flo
Aim of the experiment: Ligation of F187+F268 and F188+F268
Procedure:
Ligation batch for F187+F268, vector:insert = 1:3
volume
reagent
0.527 µl
F187
1.406 µl
F268
15.067 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F188+F268, vector:insert = 1:3
volume
reagent
0.732 µl
F188
1.406 µl
F268
14.862 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. water instead of insert
Ligation was performed at room temperature for 1 h
[Expand] Picking of transformed Plasmid E. coli XL1 blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Investigator: Louise
Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Procedure:
Picking and overnight culture after standard laboratory's protocol. (6 µl Cam, 6 ml LB-medium)
1 colony was picked for every transformation product.
[Expand] Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Inoculation of 50 ml LB meida wirh E. coli XL1 blue transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Investigator: Ingmar
Aim of the experiment: Inoculation of Midiprepcultures of E. coli XL1 blue transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Procedure:
1 ml of each of the 6 ml LB Miniprepcultures prepared by Louise was used to inoculate 50 ml Lb media with 50 µl Cam. Incubation overnight in a Minitron shaker at 37 °C and 160 RPM
Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with ligation products P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Procedure:
Ligation products were labelled B to V (in the same order as in Aim of the experiment, above)
Two clones for every ligation product except for H, L and O
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P650 to P688.
[Expand] Preparation of media for competent cells
Investigator : Johanna, Louise
Aim of the experiment:Preparation of media for competent cells
Procedure:
Picking of one colony of overnight stem culture of non competent XL1-Blue E.Coli in 50 ml LB-Medium without antibiotics (under the sterile bench)
this was incubate overnight at 37 °C
Batch for 1l of a 0.1 M MgCl2-solution
amount
reagent
20.33 g
MgCl2 (M=203.3 g/mol)
1 l
ELGA H2O
Batch for 500ml of a 50 mM CaCl2-solution
amount
reagent
3.67 g
CaCl2 (M=147.02 g/mol)
500 ml
ELGA H2O
Batch for 50ml of a 50 mM CaCl2-solution, 15% v/v Glycerin
amount
reagent
0.368 g
CaCl2 (M=147.02 g/mol)
0.458 g
Glycerin (density=1.26)
50 ml
ELGA H2O
Autoclave each bottle of solution and leave each in a 4°C room.
[Expand] Transformation of E. coli XL1-Blue with P496, P661, P629
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with P496, P661, P629.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268 and F188+F268
Investigator: Rosario
Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268 and F188+F268
Procedure:
Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
3 colones were picked for every transformation product, 2 for F188+F268 and F187+F268.
[Expand] Ligation of F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267
Investigator: Jeff
Aim of the experiment: Ligation of F230+F262 (SERK-SigP_EreB_Strep-tag-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F263 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyCatcher in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F264 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyTag_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F265 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F266 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3), F230+F267 (SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1 in PppActin_mMCS(insert location)_t35S_npt-cassette_pSB1C3)
Procedure:
Ligation batch for F230+F262, vector:insert = 1:3
volume
reagent
0.4 µl
F230
15.96 µl
F268
0.7 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F263
volume
reagent
0.4 µl
F230
16.6 µl
F263
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F264, vector:insert = 1:3
volume
reagent
0.4 µl
F230
5.9 µl
F264
10.7 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F265, vector:insert = 1:3
volume
reagent
0.4 µl
F230
3.6 µl
F265
13 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F266, vector:insert = 1:3
volume
reagent
0.4 µl
F230
2.5 µl
F266
14.1 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F267, vector:insert = 1:3
volume
reagent
0.4 µl
F230
9.8 µl
F267
6.8 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. water instead of insert.
Ligation was performed at 16 °C overnight.
[Expand] Midiprep and preparative digestion of cultures with E. coli XL1 blue transformed transformed with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Investigator: Ingmar & Louise
Aim of the experiment: Production of DNA for moss transformations with P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Procedure:
The preparation of plasmid DNA was executed according to the user manual provided by Qiagen. The final DNA pellet was dissolved in 500 µl EB buffer.
The plasmid DNA was linearized with EcoRI HF at 37 °C overnight in batches with the following composition:
Component
Volume [µl]
EcoRI HF
5
DNA
500
CutSmart
56
Sunday, August 25th
[Expand] Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)
Investigator: Johanna, Christopher, Louise
Aim of the experiment: Preparation of competent cells according to the CaCl 2-method (Cohen et al.,1972)
Procedure:
Add Tetracyclin (ratio: 1:1000) to the 2YT-medium
Measure the OD550 of it until OD550=0.5
Put the culture in Falcon tubes (leaving on ice)
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 40 ml 0,1 M MgCl2 solution after removing the supernatants
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 20 ml 50mM CaCl2 solution after removing the supernatants
Incubate the falcon tubes on ice for 30 min
Centrifuge the falcon tubes by 5000 rpm, 4°C, 10 min
Resuspend the falcon tubes each with 2 ml 50mM CaCl2, 15 % v/v Glycerin solution after removing the supernatants. From this step everything needs to be carry out in a 4°C room.
Mix the solutions of each falcon tube and transfer the content in eppis (each with 100 and accordingly 150 µl)
Store the boxes of eppis at -80 °C
[Expand] Ligation of F230+F228
Investigator: Johanna
Aim of the experiment: Ligation of F230+F228
Procedure:
Ligation batch for F230+F228, vector:insert = 1:3
volume
reagent
0.41 µl
F230(243.7 ng/µl; 5796 bp)
3.21 µl
F228(25.5 ng/µl; 1581 bp)
13.38 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. water instead of insert.
Ligation was performed at room temperature for 1 h.
[Expand]Analytical gelelectrophoresis of P742-P757,P759, P760
Investigator: Johanna
Aim of the experiment: Analytical gelelectrophoresis of P742-P757,P759, P760
Procedure:
Procedure of a mixture: 300 µl H2O + 50 µl DNA loading buffer (10x)
8 µl of DNA loading buffer (10x) were added to 1 µl Plasmid and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
P748
P749
P750
P751
P752
P753
Result:
as expected
as expected
as expected
as expected
as expected
as expected
Lane:
2log DNA ladder
P754
P755
P756
P757
P759
P760
Result:
as expected
as expected
as not expected
as not expected
as not expected
as not expected
Lane:
2log DNA ladder
P742
P743
P744
P745
P746
P747
Result:
as expected
as expected
as expected
as expected
as expected
as expected
[Expand]Transformation of E. coli XL1-Blue with ligation product F230+F228
Investigator: Johanna
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F228.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Miniprep of ligations F187+F268 (PIF3 + fret system) and F188+F268 (PIF6 + fret system) and analytical digest with EcoRI & PstI
Investigator: Jeff
Aim of the experiment: Obtaining ligated plasmids F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES + sYFP_G_TEV-site_G_cYFP) (= P761 and P762) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES + sYFP_G_TEV-site_G_cYFP) (= P763 and P764)
Procedure:
Preparation of the plasmids according to the QIAGEN Miniprep Kit, elution in 50 µl
Batch for digestion with EcoRI & PstI
volume
reagent
2.5 µl
Plasmid
2 µl
Cut Smart buffer
0.25 µl
EcoRI-HF
0.25 µl
PstI-HF
15 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1 h.
1 % agarose gel with ethidium bromide, 90 V, 30 min
Ctrl: F187 and F188 respectively
Lane:
2log DNA ladder
P761 dig. EcoRI&PstI
P762 dig. EcoRI&PstI
F187
P763 dig. EcoRI&PstI
P764 dig. EcoRI&PstI
F188
Result:
-
not as expected
not as expected
ctrl
not as expected
not as expected
ctrl
[Expand] Preparative digestion of P761-P764 with EcoRI and PstI + preparative gelelectrophoresis + gelextraction
Investigator: Jeff
Aim of the experiment: Obtaining fragments of PIF3/PIF6 with fret system for ligation into expression vector
Procedure:
Reaction batch for P761-P764:
volume
reagent
20 µl
Plasmid DNA
4 µl
CutSmart Buffer (10x)
1 µl
EcoRI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 2.5 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% agarose gel.
Analytical digest shows failed ligaton, preparative digest was cancelled
[Expand] Transformation of E. coli XL1-Blue new competent cells with P649 (Retrafo)
Investigator: Louise
Aim of the experiment: Test-Transformation with new batch of competent cells
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
5 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on a new chlorampenicol plate.
The negative control of competent cells which were not transfomed with Plasmid DNA but with water, were plated on all kind of plates: Chloramphenicol, Kanamycin, Ampicillin and tetracyclin
[Expand] Troubleshooting: Repeat of ligation of F187+F268 and F188+F268 and re-picking of the 08/23 ligation
Investigator: Jeff
Aim of the experiment: Ligation of F187+F268 and F188+F268
Procedure:
Ligation batch for F187+F268, vector:insert = 1:3
volume
reagent
0.53 µl
F187
1.41 µl
F268
15.067 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F188+F268, vector:insert = 1:3
volume
reagent
0.73 µl
F188
1.41 µl
F268
14.862 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
neg. ctrl. water instead of insert
Ligation was performed at 16 °C over night
[Expand] Picking of transformed Plasmid E. coli XL1 blue with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496
Investigator: Florian
Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496.
Procedure:
Picking and overnight culture after standard laboratory's protocol. (6 µl Cam, 6 ml LB-medium)
2 colonies were picked for every transformation product.
[Expand] Alcohol precipitation and purification of linearized Mididpreps of P650, P652,P654, P656, P658, P661, P663, P666, P672, P678, P679, P682, P685, P706, P709, P625, P414, P628, P629, P630, P632, P713
Investigator: Ingmar & Louise
Aim of the experiment: Purification of linearized DNA
Procedure:
350 µl of 1M Sodiumactetat solution (pH adjusted to 5,2 with concentrated acetic acid) were added to each digestion sample.
The DNA was precipitated with 595 µl isopropanol analytical grade. Afterwards centrifugation at 15 000 xg for 30 min at 4°C.
After having removed the supernatant the DNA pellet was washed with 1 ml 70 % EtOH. Afterwards centrifugation at 15 000 xg for 30 min at 4°C.
The supernatant was removed, the samples once again centrifuged at 16 200 xg for 30 sec. and the accumulated supernatant again removed.
The pellets were air-dryed for 2 min under the maniar flow hood.
The DNA was solved in 100 µl sterile ddH2O and stored at -20 °C.
[Expand]Analytical gelelectrophoresis of F228
Investigator: Florian
Aim of the experiment: Analytical gelelectrophoresis of F228.
Procedure:
1 µl of DNA loading buffer (10x) were added to 4 µl Fragment and were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
F228
Result:
as expected
[Expand] Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol (for P713 kanamycin) plates.
Week 19
Monday, August 26th
[Expand] Miniprep of E. coli XL1-Blue transformed with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496
Investigator: Florian
Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with Retrafo of P661 (24.8.), P661 (22.8.), P629 and P496.
Procedure:
Two clones for every ReTrafo
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P765 to P772.
[Expand] Analytical digestion and gelelectrophoresis of P769-P776 with XbaI and PstI
Investigator: Johanna
Aim of the experiment: Analytical digestion and gelelectrophoresis of P769-P776 with XbaI and PstI
Procedure:
batch for digestion of P769-P776
volume
reagent
2.5 µl
Plasmid DNA
2 µl
Cut Smart buffer
0.25 µl
XbaI-HF
0.25 µl
PstI-HF
15.0 µl
ddH2O
=20 µl
TOTAL
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and were loaded on a 0.5% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 1 h.
Lane:
2log DNA ladder
P769
P770
P771
P772
P773
P774
P775
P776
Result:
as expected
as expected
as expected
as expected
as expected
as expected
as expected
as expected
[Expand] Miniprep of E. coli XL1-Blue transformed with F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713
Investigator: Florian
Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267 and P711, P712, P713.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P780 to P799.
[Expand] Analytical digestion and gelelectrophoresis of P780 – P797
Investigator: Jeff
Aim of the experiment: Analytical digestion and gelelectrophoresis of P780 – P797.
Procedure:
Mastermix for analytical digestion with EcoRI and PstI
volume
reagent
40 µl
NEBuffer 3.1
5 µl
XbaI
5 µl
PstI
300 µl
ddH2O
=350 µl
TOTAL
17.5 µl of the mastermix were mixedd together with the respective plasmids (P780 – P797).
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 0.5% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Lane:
2 log DNA ladder
P780 digested with XbaI & PstI
P781 digested with XbaI & PstI
P782 digested with XbaI & PstI
P783 digested with XbaI & PstI
P784 digested with XbaI & PstI
P785 digested with XbaI & PstI
P786 digested with XbaI & PstI
P787 digested with XbaI & PstI
P788 digested with XbaI & PstI
P789 digested with XbaI & PstI
P790 digested with XbaI & PstI
Result:
as expected
corrupt
corrupt
corrupt
as expected
as expected
as expected
as expected
as expected
corrupt
corrupt
Lane:
2 log DNA ladder
P791 digested with XbaI & PstI
P792 digested with XbaI & PstI
P793 digested with XbaI & PstI
P794 digested with XbaI & PstI
P795 digested with XbaI & PstI
P796 digested with XbaI & PstI
P797 digested with XbaI & PstI
Result:
corrupt
corrupt
corrupt
as expected
as expected
as expected
as expected
[Expand] Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268, F188+F268, F230+F228 and Retrafos of P711, P712, P713, P649
Investigator: Rosario
Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with ligation product F230+F262, F230+F263, F230+F264, F230+F265, F230+F266, F230+F267, F187+F268,F188+F268, F230+F228 and Retrafos of P711, P712, P713, P649
Procedure:
Picking and overnight culture after standard laboratory's protocol. (4 µl Cam, 4 ml LB-medium)
3 colones were picked for every ligation product (5 for F187+F268,F188+F268) , 2 for the Retrafos.
Tuesday, August 27th
[Expand] Transformation of E. coli XL1-Blue with ligation products F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2)
Investigator: Jeff
Aim of the experiment: Transformation of E. coli XL1-Blue with ligation products F187+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2) and F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand] Picking of transformed Plasmid E. coli XL1 blue with P6,P773-P776
Investigator: Johanna
Aim of the experiment: Picking of transformed Plasmid E. coli XL1 blue with P6,P773-P776
Procedure:
P773-P776: 2 Colonies were picked from chloramphenicol plates.
P6: 2 Colonies were picked from ampicilin plates.
P773-P776: Each colony was transferred into a tube containing 6 mL of LB-medium + 6 µL chloramphenicol(1000x).
P6: Each colony was transferred into a tube containing 6 mL of LB-medium + 6 µL ampicilin(1000x).
These tubes were transferred into a cell culture shaker at 37 °C and were incubated 12 h
[Expand] Miniprep of E. coli XL1-Blue transformed with P649, F230+F196, F230+262, F230+263, F230+264, F230+266, F230+267
Investigator: Rosario
Aim of the experiment: Miniprep of E. coli XL1-Blue transformed with P649, F230+F196, F230+262, F230+263, F230+264, F230+266, F230+267.
Procedure:
Miniprep was performed after manufacturer's protocol (QIAprep Spin Miniprep Kit, QIAGEN).
The resulting tubes were labeled as P800 to P809.
[Expand] Transformation of E. coli XL1-Blue with P794
Investigator: Rosario
Aim of the experiment: Transformation of E. coli XL1-Blue with P794.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates.
[Expand]Preparative digestion of P290, P387, P787 with EcoRI and PstI, and preparative digestion of P799 with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction
Investigator: Rosario
Procedure:
Reaction batch for P290/P387/P787:
volume
reagent
20 µl
Plasmid DNA
4 µl
NEB 2.1 Buffer (10x)
1 µl
EcoRI-HF (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batch for P799:
volume
reagent
20 µl
Plasmid DNA
4 µl
NEB 3.1 Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
14 µl
ddH2O
=40 µl
TOTAL
Reaction batches were incubated at 37 °C for 3 h.
Preparative gelelectrophoresis was performed at 90 V for 1 h in an 0,5% (P787) and 1% agarose gel.
The results were corrupt so the experiment was repeated
The results were corrupt so the experiment was repeated
[Expand] Analytical digestion and gelelectrophoresis of P800 – P809
Investigator: Rosario
Aim of the experiment: Analytical digestion and gelelectrophoresis of P800 – P809.
Procedure:
Mastermix for analytical digestion with EcoRI and PstI
volume
reagent
24 µl
NEBuffer 3.1
3 µl
XbaI
3 µl
PstI
180 µl
ddH2O
=210 µl
TOTAL
17.5 µl of the mastermix were mixedd together with the respective plasmids (P780 – P797).
Analytical digestion was performed at 37 °C for 1.5 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Lane:
2 log DNA ladder
P800 digested with XbaI & PstI
P801 digested with XbaI & PstI
P802 digested with XbaI & PstI
P803 digested with XbaI & PstI
P804 digested with XbaI & PstI
P805 digested with XbaI & PstI
P806 digested with XbaI & PstI
P807 digested with XbaI & PstI
P808 digested with XbaI & PstI
P809 digested with XbaI & PstI
Result:
as expected
corrupt
corrupt
corrupt
as expected
as expected
as expected
as expected
as expected
corrupt
[Expand] Retransformation of E. coli XL1-Blue with P290, P387, P787, P799
Investigator: Rosario
Aim of the experiment: Retransformation of E. coli XL1-Blue with P290, P387, P787, P799.
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates (Kanamycin for P799).
[Expand] Picking of Ligation F228+F230 (banana cytoplasm. + plasmid w/ Mini MCS)
Investigator: Rosario
Aim of the experiment:
Picking of Ligation F228+F230 (banana cytoplasm. + plasmid w/ Mini MCS)
Procedure:
6 clones picked; 4 ml LB medium w/ 4 µl Cam each
Incubation at 37 °C over night
[Expand] Picking of Ligations F188+F268 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF6_IRES_eCFP_G_TEV-site_G_SYFP2), F230+F265 (PppActin_SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag_t35S_npt-cassette), F230+F266 (PppActin_SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc_t35S_npt-cassette) and F230+F267 (PppActin_SERK-SigP_Laccase_Strep-tag-II-TEV-site-Linker_SERK-TMD_8AALinker_GFPmut1_t35S_npt-cassette)
Investigator: Jeff
Aim of the experiment: Picking of Ligations F188+F268, F230+F265 (from 08/25 and from 08/26), F230+F266 (from 08/25 and from 08/26) and F230+F267 (from 08/25 and from 08/26)
Procedure:
6 clones F188+F268
3 clones each of F230+F265, F230+F266 and F230+F267 from 08/25
3 clones each of F230+F265, F230+F266 and F230+F267 from 08/26
4 ml LB medium w/ 4 µl Cam each
Incubation at 37 °C over night
[Expand] Miniprep of P6 (pGAL in pTUM100)
Investigator: Jeff
Aim of the experiment: Preparation of P6 plasmid DNA
Procedure:
Preparation according to QIAGEN Miniprep Kit, elution in 50 µl EB buffer each
plasmid
c (ng/µl)
P810
384.2
P811
134.1
[Expand] Preparative digestion of P810 and P811 (pGAL in pTUM100) with with XbaI and PstI + Preparative Gelelectrophoresis + Gelextraction
Investigator: Jeff, Ingmar
Procedure:
Reaction batch for P810 and P811
volume
reagent
25 µl
Plasmid DNA
5 µl
NEB 3.1 Buffer (10x)
1 µl
XbaI (20 U/µl)
1 µl
PstI (20 U/µl)
18 µl
ddH2O
=50 µl
TOTAL
Incubation at 37 °C for 2.5 h.
Preparative gelelectrophoresis at 90 V for 30 min on 1% agarose gel.
[[File:|500px]]
[Expand] Ligation of F230+F273, F230+F274, F230+F275, F276+F277
Investigator: Jeff
Aim of the experiment: Ligation of F230+F273, F230+F274, F230+F275, F276+F277.
Procedure:
Ligation batch for F230+F273, vector:insert = 1:3
volume
reagent
0.40 µl
F230 (243.7 ng/µl; 5796 bp)
2.2 µl
F273 (31.7 ng/µl; 1327 bp)
14.4 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F274, vector:insert = 1:3
volume
reagent
0.40 µl
F230 (243.7 ng/µl; 5796 bp)
1.8 µl
F274 (46.5 ng/µl; 1597 bp)
14.8 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F230+F275, vector:insert = 1:3
volume
reagent
0.40 µl
F230 (243.7 ng/µl; 5796 bp)
2.1 µl
F275 (166.9 ng/µl; 6751 bp)
14.5 µl
ddH2O
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
Ligation batch for F276+F277
volume
reagent
4.3 µl
F276 (243.7 ng/µl; 3978 bp)
12.7 µl
F277 (166.9 ng/µl; 72 bp)
2 µl
T4 ligase buffer (10x)
1 µl
T4 ligase (1 U/µl)
=20 µl
TOTAL
For neg. ctrl. water was used instead of insert.
Ligation was performed at room temperature for 1 h and then put on 16 °C for the next shift to transform the ligation products.
Wednesday, August 28th
[Expand] Transformation of E. coli XL1-Blue with ligation products F230+F273 (Ig-Kappa-SigP_EreB + Plasmid with Mini-MCS), F230+F274 (IgKappa-SigP_Laccase + Plasmid with Mini-MCS), F230+F275 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2 + Plasmid with Mini-MCS) and F276+F277 (pBad + digested pGAL)
Investigator: Louise
Aim of the experiment:Transformation of E. coli XL1-Blue with ligation products F230+F273 (Ig-Kappa-SigP_EreB + Plasmid with Mini-MCS), F230+F274 (IgKappa-SigP_Laccase + Plasmid with Mini-MCS), F230+F275 (PhyB_36AAlinker_C-TEV_IRES_N-TEV_36AAlinker_PIF3_IRES_eCFP_G_TEV-site_G_SYFP2 + Plasmid with Mini-MCS) and F276+F277 (pBad + digested pGAL).
Procedure:
CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.
10 µl of DNA were added to the tubes containing 150 µl of competent cells and gently mixed
30 min incubation on ice
5 min. heat shock at 37 °C
Adding of 1 ml LB-medium to each tube.
Incubation for 90 min at 37 °C in the 180 rpm cell-culture shaker.
The cell suspensions were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.
The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on new chlorampenicol plates (having F230 as backbone) and kanamycin plates (having pBad as backbone).
[Expand] Miniprep of ligation products F265+F230, F228+F230, F267+F230, F266+F230 and F268+F188
Investigator: Louise
Aim of the experiment:
Procedure:
Preparation according to QIAGEN Miniprep Kit, elution in 50 µl EB buffer each
The Plasmids were labelled as following:
ligation product
label
F265+F230 clones 1a-3b
P812-P817
F268+F188 clones 1-6
P818-P823
F228+F230 clones 1-5
P824-P828
F267+F230 clones 1a-3b
P829-P834
F266+F230 clones 1a-3b
P835-P840
[Expand] Analytical digestion and gelelectrophoresis of P812-P840
Investigator: Louise
Aim of the experiment:Analytical digestion and gelelectrophoresis of P812-P840 .
Procedure:
Batch for digestion with XbaI and PstI
volume
reagent
2.5 µl
Plasmid DNA
2.0 µl
NEBuffer 3.1
0.25 µl
XbaI
0.25 µl
PstI
15 µl
ddH2O
=210 µl
TOTAL
Analytical digestion was performed at 37 °C for 1 h.
After incubation 2 µl of DNA loading buffer (10x) were added to the reaction batches and 10 µl were loaded on a 1% agarose gel.
Analytical gelelectrophoresis was performed at 90 V for 60 min.
Lane:
2 log DNA ladder
P812 digested with XbaI & PstI
P813 digested with XbaI & PstI
P814 digested with XbaI & PstI
P815 digested with XbaI & PstI
P816 digested with XbaI & PstI
P817 digested with XbaI & PstI
P818 digested with XbaI & PstI
P819 digested with XbaI & PstI
P820 digested with XbaI & PstI
P821 digested with XbaI & PstI
P822 digested with XbaI & PstI
Result:
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
Lane:
P823 digested with XbaI & PstI
2 log DNA ladder
P824 digested with XbaI & PstI
P825 digested with XbaI & PstI
P826 digested with XbaI & PstI
P827 digested with XbaI & PstI
P828 digested with XbaI & PstI
P829 digested with XbaI & PstI
P830 digested with XbaI & PstI
P831 digested with XbaI & PstI
P832 digested with XbaI & PstI
P833 digested with XbaI & PstI
Result:
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
TBD
Lane:
P834 digested with XbaI & PstI
2 log DNA ladder
P835 digested with XbaI & PstI
P836 digested with XbaI & PstI
P837 digested with XbaI & PstI
P838 digested with XbaI & PstI
P839 digested with XbaI & PstI
P840 digested with XbaI & PstI
Result:
TBD
TBD
TBD
TBD
TBD
TBD
TBD
Thursday, August 29th
[Expand] Midiprep cultures from Miniprep cultures P815 and P835
Investigator: Jeff
Aim of the experiment: Midiprep culture from Miniprep cultures P815 (SERK-SigP_SpyCatcher_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_NanoLuc_SpyTag + Plasmid mit Mini MCS) and P835 (SERK-SigP_SpyTag_Strep-Tag-II-TEV-site-linker_SERK-TMD_8AAlinker_GFPmut1_IRES_IgKappa-SigP_SpyCatcher_NanoLuc + Plasmid mit Mini MCS)
Procedure:
50 ml LB medium w/ 50 µl chloramphenicol stock (1:1000)
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