Team:Goettingen/NoteBook w13

Harvesting of cells for qRT-PCR (promoter characterization)

measurement of OD600nm again

-          harvesting of another 1 ml sample (OD600nm= 2.42 to 2.91), as described on 28.8.13

-          calculation of growth curves → see excel sheet in dropbox or department server folder

RNA extraction from E. coli

with kit: Qiagen RNeasy® Plus Mini Kit

-          clean bench and pipets with ethanol

-          prepare RLT buffer with 2-mercaptoethanole (1 ml RLT buffer → 10 µl 2-mercaptoethanol); here: for 18 samples, 20 ml RLT buffer were supplied with 200 µl 2-mercaptoethanol (in other Bacillus-lab in the hood)

-          prepare lysis buffer: 1xTE buffer + 0.5 mg/ml lysozyme in RNase free water (here: 400 µl 10x TE (sterile) + 100 µl 20 mg/ml lysozyme + 3.5 ml RNase free water→ 4 ml lysis buffer)

-          set one heating block to 70 °C and pre-heat RNase free water for elution (100 µl for elution of 1 sample)

-          set another heating block to 37 °C for lysis

a) Lysis: add 200 µl lysis buffer to E. coli pellet, resuspend properly and incubate for 10 min at 37 °C;add 1 ml RLT supplied with 2-mercaptoethanol to the suspension and centrifuge for 5 min at 13 000 rpm, RT (I made a mistake and loaded the mixture directly on the gDNA columns (for all exponential phase and for all stationary phase samples except P8 C1 → so I pipetted everything back into the remaining mixture (some solution has also passed the membrane, I pipetted this part back, as well) and centrifuged, then I loaded new columns with the supernatant after centrifugation…)

b) removal of gDNA: load supernatant in 700 µl steps on violet columns and centrifuge at 13 000 rpm, RT, 1 min→ collect FLOW-THROUG in 2 ml epi tube (column binds gDNA, but NOT RNA → RNA is in FLOW-THROUG!!!)

c) binding of RNA: add more or less equal volume (here: 1 ml) 70 % EtOH (cooled) to flow-through, mix by pipetting and vortexing, then load 700 µl of mixture on pink columns (pink columns bind RNA), centrifuge 1 min, RT, 13 000 rpm, discard flow-through; repeat loading + centrifuging until the whole mixture is used up

d) washing: → now, one has to discard the flow-through…

d1) load 700 µl RW1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

d2) load 500 µl RPE1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

d3) load again 500 µl RPE1 buffer onto column, centrifuge at 13000 rpm, RT, 1 min

e) drying of column: centrifuge column in emptied collection tube 1 min, 13000 rpm, RT

f) elution: place column in fresh epi tube (RNase-free) elute RNA with 50 µl pre-heated (70°C) RNase-free water, incubate tubes at 70 °C for 5 min, then spin down at 13000rpm for 1 min, RT (here: sample “exp. 8” was eluted with 100 µl by mistake…)

g) measure concentration with NanoDrop (set it to RNA!); Blank: HPLC water (no difference to RNase free water…)

NanoDrop concentrations from today:

Abbreviations: exp. = exponential phase (sample taken at OD600nm= ca. 0.6); stat. = stationary phase (sample taken at OD600nm> 2); ON = sample taken after ON incubation (OD600nm> 2); 1 = Part 1 C1; 2-1 = Part 2 C1; 2-2 = Part 2 C2; 3 = Part 3; 4 = Part 4; 8 = Part 8

→ or ON samples, a peak at ca. 225 nm was observed

→ in general the A260nm/A280nm seem to be quite low…

samples stored at -80 °C in green plastic box

Re-transformation of Promoter and RBS Part plasmids 

-          for characterization, 3 biological replicates should be done

-          so far, with P1 C1, P2 C1 and C2, P3 C1 and P4 C1 and P8 C1, we have one biological replicate. The plasmids from the “proper” clones (not P2 C1 plasmid!) will be re-transformed into DH5α and 2 clones will be picked and further analyzed.

plasmid solutions used:

Part 1 C1 plasmid (84.6 ng/µl), 7.6.13

Part 2 C2 plasmid (120.6 ng/µl)

Part 3 C1 plasmid (151.0 ng/µl), 7.6.13

Part 4 C1 plasmid, 2.7.13

Part 8 C1 plasmid (113.9 ng/µl), 11.7.13

-          1 µl plasmid solution was added to DH5α comp. cells, then transformation was done according to methods-folder-protocol

-          a neg. control was included (1 µl sterile dH2O added to comp. cells)

-          500 µl supernatant were removed after centrifugation

-          plating on LBAmp plates and incubation at 37 °C

Colony PCR for DarR reporter system clones

Transformation from 28.8.13:

neg. control: no colonies on 50 µl plate, nor on rest-plate

w/o insert control: no colonies on 50 µl plate, but 1 or 2 on rest-plate → bigger one picked for Colony PCR, since regarding the smaller one, I was not sure if it was really a clone or a bubble…; inoculation of purple Colony PCR tube (termed R)

with A or B insert: no colonies on 50 µl plate, but several on rest-plate  → 12 clones picked each for Colony PCR: A → blue PCR tubes; B → green PCR tubes, termed 1 – 12

Colony PCR:

-          colony PCR was done as described previously (10.7.13)

-          an additional control was used: plasmid DarRrev-Termrev C3 plasmid, which was digested to generate the vector (plasmid solution form 23.8.13, 229.7 ng/µl, dilution 1:25 → ca. 9 ng/µl), yellow PCR tube, termed P

-          primers: iGEM_38 (VF2) and iGEM_39 (VR)

-          30x MasterMix prepared, distribution of 15 μl to 26 tubes, addition of 1 μl plasmid dilution or inoculation with clones after streak-out on master plates → master plates incubated over day at 37 °C

-          protocol: elongation time increased to 4:30 min, since almost 2 kb insert in positive clones

Gel run for Colony PCR (by Katrin G.) 

-          2x 1% agarose-1xTAE gel

-          addition of 5 µl 5x LD to 25 µl PCR reaction

-          loading of 5 µl on gel

-          loading of 3 µl 2 log ladder as a marker

-          run

-          EtBr staining + destaining in water

-          UV detection

Gel 1 for DarR reporter system A:

Loading: Marker/ DarRrev-Termrev C3 plasmid/Re-ligand/C1/C2/C3/C4/C5/C6/C7/C8/C9/C10/C11/C12/Marker

Gel 2 for DarR reporter system B:

Loading: Marker/C1/C2/C3/C4/C5/C6/C7/C8/C9/C10/C11/C12/ DarRrev-Termrev C3 plasmid/ Re-ligand/Marker

→ The expected band of ca. 2 kb is not seen, but the band of re-ligated vectors was observed for re-ligand and plasmid control (> 1 kb, as expected) and also for B C5→ since this band is not seen for all other clones, but no 2 kb and since there are bands at smaller bp  which are not observed for the re-ligand, it could be that the elongation time was still too short/that Taq hat some problem to amplify insert of positive clones → all clones w/o re-ligand band could be considered as positive clones → inoculation of A C1 – C5 and B C1 – C4 and C6 for MiniPrep and TestRD, to see if they are really positive clones.

Reactions thrown away

-          plates stored at 4 °C (big fridge)

Inoculation

-          Inoculation of DarR reporter system clones A C1 – C5 and B C1 – C4 and B C6 in 4 ml LBCm (from master plates)

-          Incubation ON at 37 °C, 200 rpm

-          Plates stored at 4°C, big fridge

Plate reader assay (characterization of promoter clones)

-          plate reader stopped

-          data analysis… (see excel sheet in dropbox/department server) 

-          For the growth curves, the mean of the OD600nm values from the technical replicates was calculated. The error bars indicate the standard deviation.

-          For the RFP/OD600nm curves, the RFP values (in RFU) were normalized to OD600nm. The mean was calculated using the normalized values from the technical replicates. The error bars indicate the standard deviation.

-          RFP fluorescence had large errors during initial log phase (ca. 0 – 3 h) and, for P3 and P4, during remaining log phase and stationary phase, but OD600nm not

-          cells showed logarithmic growth in first 5 h, then, growth curve flattened indicating that stationary phase was reached; over the time, cell amount seemed to decrease slightly

-          in the beginning (first 3 h (up to 5h) of incubation, exponential growth phase), the values for normalized RFP fluorescence varied strongly; this might be caused by the low OD600nm(https://2012.igem.org/Team:LMU-Munich/Data/Anderson). During late log phase/beginning of stationary phase at ca. 6 – 7 h, the fluorescence increased suddenly and strongly à fluorophores of RFP might need oxygen for maturation/formation à during log phase, cells grew strongly and might have used oxygen necessary for RFP maturation/fluorescence. During stationary growth, the fluorescence is relatively constant correlating with the constant OD600nm.

-          P1 C1, P2 C2, P3 C1 and P4 C1 seemed to differ significantly from empty-vector control P8 C1 regarding RFP fluorescence: the RFP fluorescence was higher during stationary phase for all promoters. àRFP seemed to have been expressed and matured in higher amounts for Promoter 1, 2, 3 and 4 during stationary phase.

-          relative strength of promoters: Promoter 1< Promoter2<<Promoter 4 < Promoter 3

-          P2 C1 did not differ significantly from empty-vector control P8 C1à P2 C1 possibly does not contain RFP/ Promoter.

“Drop”-Experiment: Photographs taken

Pictures:

RiboA on Gp1013 without (left), and with (right) IPTG

RiboA on p172 without (left) and with (right) IPTG

Results/Discussion:

There are no visible differences between the controls and the RiboA on top of induced (and therefore cdiAMP-producing) cells.

Also, the cells in the agar are not really homogenously distributed but grow in clones. The holes in the fluorescent clones seem to be clones of the cells, which are supposed to be inside the plates. The next plates need to be made with a new protocol addressing this problem.

Reinocculated the CFP-religation clones, the DAC, and the Empty Vector clone

Procedures with these clones for tomorrow:

CFP-Religation C1 and C2:        

1.Inocculation of 10ml cultures for Glycerin Stocks

2.Inocculation of Synchronization Culture (see DAC and Empty and RiboA and CFP clones)

3.Cryostocks

4.Minipreps and Sequencing

DAC and Empty Vektor: Competition Experiment together with RiboA and the CFP clones. Therefore Synchronization culture tomorrow morning.

Picked and inocculated 5 Clones from the Retrafo of RiboA C5.

Procedures with these clones:

1.Inocculation of Synchronization culture with DAC and Empty vector (not together, just at the same time) for the competition exp. and a fluorescence screen over time

2. Cryostocks

3. Minipreps? 

The names of the clones are now RiboA C5.3.1 – C5.3.5

Sequencing results: G2L, Promoter 3rev in part 6.2

-          for all plasmids, the sequence stops at the same region as for the seqlab sequences

-          DMSO and denaturing didn’t help…

→ sequencing of Promoter3rev in pSB1C3 with VF2 and VR (from behind insert) and sequencing of Promoter3rev in part 6.2 with reverse qRT-PCR GFP primer…

Transformation of ligations from 27.8.13 (DarR reporter system)

-          comp. cell strain: XL1-Blue

-          for neg. control, 20 μl sterile dH2O were added to the cells

-          whole ligation reactions were transformed

-          removal of 500 μl supernatant after centrifugation

-          plating on LBCm plates

-          incubation ON at 37 °C (started at ca. 11:20 a.m)

Harvesting of cells for qRT-PCR analysis and plate reader assay (promoter clones)

-          measuring of OD600nm of ON cultures in 1:20 dilution of LB and calculation of actual OD600nm→ OD600nm in ON cultures varied from 3.5 to 3.84 → inoculation of synchronization pre-culture: 4 ml LBAmp + 50 μl ON culture, incubation at 37 °C at ca. 200 rpm for > 2,5 h (in between, the OD600nm was measured and the cells kept on ice → don’t keep cells on ice!!! keep them on bench!)

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm 

-          preparation of plate reade titer plate (170 µl culture) and 100 ml Erlenmeyer flask w/o baffles (15 ml culture), both cultures: OD600nm = 0.05

a) OD600nm ON culture

b) measurement of synchronization culture in between

c) measurement of synchronization culture before main culture and titer plate preparation

The volumes indicated in the table were added to 160 µl or 15 ml LBAmp, respectively

*calculation: Volume (of new culture) * OD600nm (of new culture)/OD600nm (old culture) = Volume (old culture, needed for preparation of new culture)

qRT-PCR: Harvesting of Cells

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm→ inoculation of main cultures: 15 ml LBAmp, OD600nm =0.05 (approx.)

-          incubation at 37 °C, 200 rpm

-          growth curve → measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm over time, plot of Log2 (OD600nm) against time (in h)

-          harvesting of 2 ml culture at an OD600nm = ca. 0.6 (log phase) by centrifuging for 3 min at 14.8 rpm, 4 °C, then supernatant taken off completely and cell pellet frozen in liquid nitrogen, storage at – 80 °C, harvesting of 1 ml culture at an OD600nm = ca. 2 (stationary phase), as described before

-          after harvesting 1 ml, the cells were incubated ON (in total ca. 20 h)

Plate Reader assay

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm→inoculation of plate reader wells: 170 μl LBAmp, OD600nm = 0.05 (approx)

-          incubation at 37 °C, strong shaking

-          measuring of OD600nm against LBAmp as blank

-          measuring of RFP fluorescence (excitation: 555 nm; emission: 583 nm)

-          run over day and ON

-          pipetting scheme (I made a mistake and pipetted a higher volume of P4 C1 culture in row 6, correct volume of P4 C1 culture was added in row 7)

RiboA Cells inocculated yesterday did not grow except for C5.8 from yesterday´s overday-culture picked from the plate from the 21.8.

Discussion: RiboA C5.8 will be our Antibiotic Detector!

“Drop”-experiment with the DAC and empty vector control plates made by Jan yesterday

4 plates:

1x Gp1013 without IPTG

1x Gp1013 with IPTG

1x p172 without IPTG

1x p172 with IPTG

On each plates 3 drops of RiboA:

1x undiluted overnight culture

1x 1:10 diluted overnight culture

1x undiluted overnight culture smeared over one half of the plate

Plates incubated at 37°C overnight

Preparation of RiboA C5.8 cells for plates as already made by Jan yesterday

Retrafo of RiboA C5.8 from Mini-prep from (DATE?)

Inocculation of the DAC cells, the empty vector control for the second Drop”-experiment and the CFP w/o insert religation control as a control for our detector

Preparation of 2x 500 ml LBAmp plates (stored in cold room, “Görke” shelf)

Test gel for DarRrev-Termrev C3 vector after 2nd digest (with PstI)

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection 

loading: Marker/uncut DarRrev-Termrev C3 plasmid/ RD DarRrev-Termrev C3 vector

digest still partial (expected band at ca. 3kb is observed, but also a weak band at ca. 6 – 8 kb of uncut plasmid) → dephosphorylation of vector with AP to avoid self-ligation

Preparation of LB media

-          1x 300 ml broth LB w/o antibiotics

-          1x 500 ml LBCm plates

Dephosphorylation of DarRrev-Termrev C3 vector cut with SpeI and PstI

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1.5 h at 37 °C (PstI forms 3’ overhang, but dephosphorylation often incomplete → incubation for more than 1 h)

Preparation of CryoStocks of clones inoculated yesterday

DMSO cryostocks prepared as described before (13.08.2013)

for all clones inoculated on 26.8.13:

-          part 1 C2

-          part 2 C3

-          part 3 C2 and C3

-          part 4 C2 and C3

-          DarRrev-Termrev in pSB1C3 C3

-          part 6.4 A C3

-          part 6.4 B C2 and C5

-          Promoter1rev in pSB1C3 C4

-          Promoter3rev in pSB1C3 C5

Plasmid Mini Preparation 

-          harvesting of all cultures inoculated yesterday

-          but MiniPrep only for the following clones:

DarRrev-Termrev in pSB1C3 C3

part 6.4 A C3

part 6.4 B C2 and C5

Promoter1rev in pSB1C3 C4

Promoter3rev in pSB1C3 C5

-          all other clones were harvested and the cell pellets stored in to-do-box (it could be that we won’t need the plasmids…):

part 1 C2

part 2 C3

part 3 C2 and C3

part 4 C2 and C3

-          elution 1x with 30 μl HPLC water (pre-warmed), incubation at 50 °C for 2 min

-          NanoDrop concentration measurement:

Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection 

loading: Marker/DarRrev-Termrev C3 plasmid/ part 6.4 A C3 uncut plasmid (from today’s prep)/ part 6.4 A C3 vector (E + X)/part 6.4 B C2 uncut plasmid (from today’s prep)/ part 6.4 B C2 vector (E + X)/ part 6.4 B C5 plasmid/Marker

→ for vector digests, the expected bands were obtained (ca. 3 kb), but the digestes are still slightly incomplete (weak 2 kb band) → dephosphorylation with AP

→ plasmids purified today look normal

Dephosphorylation of part 6.4 A and part 6.4 B vector with AP

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1 h at 37 °C (EcoRI and XbaIform5’ overhang, but dephosphorylation often incomplete → incubation for more than 15 min)

Test RD of Promoter1rev and Promoter3rev in pSB1C3 

2 μl plasmid (from today’s purification, ca. 200 – 300 ng plasmid)

1 μl EcoRI FD

1 μl PstI FD

1 μl 10x FD Green buffer

5 μl dH2O

in total: 10 μl

-          both reactions were pipetted individually

-          incubation for 1 h at 37 °C

Gel run: Test RD of Promoter1rev and Promoter3rev in pSB1C3

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 5μl RD reaction

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection

Loading: marker/uncut Promoter1rev C4 plasmid/ RD Promoter1rev C4 plasmid/ uncut Promoter3rev C5 plasmid/ RD Promoter3rev C5 plasmid

→ both digests are only partial

→ but the expected bands were obtained: 112 bp Promrev + 2x20 bp prefix/suffix = 152 bp and ca. 2 kb of pSB1C3 backbone

→ sequencing of both plasmids

PCR clean-up of vectors (part 6.4 A and part 6.4 B vector; DarRrev-Termrev C3 vector) after AP treatment

-          with Qiagen PCR purification kit

-          addition of 500 μl PB buffer

-          elution with 30 μl HPLC water, incubating for 2 min at 50 °C, centrifugation by mistake for 2 min at 13 000 rpm

-          NanoDrop concentration measurement:

Plates Prepared for the Drop experiments

4 plates:

1x Gp1013 without IPTG

1x Gp1013 with IPTG

1x p172 without IPTG

1x p172 with IPTG 

As follows:

25ml sterile water were filled intotwo 50ml falcons. Also two of the 300µl Glycerin stocks (Gp1013 and p172) prepared earlier were added to the water in the dedicated falcons. The falcons were inverted several times. 2x LB medium was prepared by Katrin Gunka and melted in the microwave. It was added to the 25ml water rather hot to the total volume of 50ml. The falcons were inverted a few times and then one 25ml plate was poured from each falcon. Then, very fast, where needed, 25µl IPTG 1M (end concentration 1mM) were added.

Inoculation of Promoter clones for Plate Reader Assay and RT-PCR analysis

-          inoculation of

part 1 C1

part 2 C1

part 2 C2

part 3 C1

part 4 C1

part 8 C1 (as empty vector control, AmpR, but no RFP, only RBS)

-          in LBAmp from cryostocks:(scratch some frozen E.coli cells from culture tube with a yellow pipet tip and transfer them directly in LBAmp)

-          incubation ON at 37 °C at 200 – 210 rpm

Sequencing results from 23.8.13

9 – DarRrev-Termrev C2 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

10 – DarRrev-Termrev C2 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

11 – DarRrev-Termrev C3 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

12  - DarRrev-Termrev C3 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

13 – part6.4 B C2 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

14 – part 6.4 B C5 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

15 – part 6.4 A C2 + VF2 → RBSrev is not inserted

16 – part 6.4 A C3 + VF2→ RBSrev is inserted in the desired orientation, but a part of pSB1C3 is missing at the EcoRI restriction site (but EcoRI restriction site is present) and a G of NotI restriction site in prefix is missing…

17 - part 6.4 A C5 + VF2→ RBSrev is not inserted

18 – part 6.4 A C8 + VF2→ RBSrev is not inserted 

Plan:  sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI (one would get rid of missing/erroneous regions) and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2

Plates for C1 – C3 of parts 1 – 4

-          today, the part 2 clones C2 and C3 are slightly pink, while C1 is white as C1 and C2 of part 1 (C3 of part 1 never existed…)

-          clones of part 3 and part 4 are very pink

-          pictures were taken:

Restriction digests

a) DarRrev-Termrev in pSB1C3; C3 with EcoRI and SpeI 

3 μl SpeI FD

3 μl EcoRI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

23 μl dH2O

in total: 40 μl

b) part 6.4 A C3 and part 6.4 B C2 with EcoRI

4μl EcoRI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

17μl dH2O

in total: 40 μl

Cloning strategy changed -  suddenly new idea occurred to Katrin and me: sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2; but to avoid a waste of material, the above mentioned reactions were incubated as well and stored at – 20°C in DarR box (in addition, they can serve as a control for part 6.4A, to see, if EcoRI site is really there!)

c) DarRrev-Termrev in pSB1C3; C3 with SpeI

4μl SpeI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

25μl dH2O

in total: 40 μl

d) part 6.4 A C3 and part 6.4 B C2 with XbaI and PstI

3μl PstI FD

3 μl XbaI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

15μl dH2O

in total: 40 μl

→ all reactions incubated for 1.5 h at 37 °C (incubation time decreased since missing part of vector in plasmid part 6.4 A C3 might result from EcoRI FD overdigest within 2 h incubation time…)

Test Gel run for Restriction digests (see above)

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection

Gel:

Loading: Marker/ part 6.4 A C3 uncut/ part 6.4 A C3 RD EcoRI/ part 6.4 A C3 RD XbaI + PstI/ part 6.4 B C2 uncut/ part 6.4 B C2 RD EcoRI/ part 6.4 B C2 RD XbaI + PstI/DarRrevTermrev C3 uncut/ DarRrevTermrev C3 RD EcoRI + SpeI/ DarRrevTermrev C3 RD SpeI/Marker

→ both digests of part 6.4 A seemed to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of part 6.4 B seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of DarRrev-Termrev seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for EcoRI/SpeI double digest were obtained, i.e. ca. 0.8 kb and ca. 2 kb band; expected band for SpeI single digest, i. e. ca. 2.8 kb band, was also obtained)

-          purification of inserts (except for DarRrev-Termrev insert, stored unpurified at – 20 °C in To-do-box) by gel ex and purification of vectors by PCR clean-up

Purification of vectors from today by PCR clean-up (DarRrev-Termrev vector/part 6.4 A/B vector) after first round of digest

-          with Qiagen PCR purification kit

-          500 μl PB buffer were added to the samples

-          elution with 30 μl pre-warmed HPLC water, incubation for 2 min at 50 °C

-          further digest

Second round of restriction digest to generate vectors for ligation

a) DarRrev-Termrev vector previously cut with SpeI

30 μl linearized plasmid

2 μl dH2O

4 μl PstI FD

4 μl FD buffer 10x

in total: 40 μl

b) part 6.4 A/B vector previously cut with EcoRI

30 μl linearized plasmid

2 μl dH2O

4 μl XbaI FD

4 μl FD buffer 10x

in total: 40 μl

-          incubation of all three reactions at 37 °C for 1.5 h

-          samples stored at – 20 °C in to-do-box

Purification of part 6.4 A/B inserts by gel extraction

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of entire RD reaction (ca. 37 μl supplied with 7μl 5xLD)

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 85 V

-          brief EtBr staining + brief destaining in water

-          short UV detection, then gel extraction as described on 25.6.13

gel after gel ex:

loading: Marker/ uncut part 6.4 A C3/ - /RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/-/RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/uncut part 6.4 B C2/ Marker

Inoculation of clones for CryoStocks and MiniPrep for Test RD/Sequencing

inoculation of

-          part 1 C2

-          part 2 C3

-          part 3 C2 and C3

-          part 4 C2 and C3

→ in 4 ml LBAmp

→ all for cryo stocks and MiniPrep

inoculation of

-          DarRrev-Termrev in pSB1C3 C3 → for cryostock and MiniPrep

-          part 6.4 A C3 → for cryostock and MiniPrep

-          part 6.4 B C2 and C5 → for cryostock and MiniPrep

-          Promoter1rev in pSB1C3 C4 → for cryostock, MiniPrep, test RD and sequencing

-          Promoter3rev in pSB1C3 C5 → for cryostock, MiniPrep, test RD and sequencing

→ in 4 ml LBCm 

incubation ON at 37 °C, 200  - 210 rpmpurification: no isopronol used since fragments ca. 1 kb; each sample (part 6.4 A or part 6. 4 B) was distributed onto two colums, DNA from each column was eluted with 30 μl pre-warmed HPLC water (incubation of sample for 2 min at 50 °C. The DNA from both columns of one sample was collected in the same tube.

-          NanoDrop concentration measurement:

-          sample stored in To-do-box

Inocculation of Ribo A C5.6 and C5.8 from cryo stocks in LB medium containing Cm

Inoccultion of DAC (GP1013) and the empty vector (pGP172) from cryo stocks in LB medium containing Amp

both stored at 30°C over night in the shaker