Team:Heidelberg/Templates/DelH week18

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Contents

26-08 - 01-09-13

Generation of DelH Plasmid pHM05 26-08

Overview on Fragments

Fragment Concentration [ng/µl] Date
G0 6.4 22-8
G1/2a 8.5 22-08
G2b 18.6 09-08
BB 40.6 22-08
TetR 132.1 26-08


Gibson Assembly

Mix name DelH-G0 DelH-1/2a DelH-G2b BB17 TetR (1:10) GibsonMasterMix Final volume
0 8.5 µl - - 0.5 µl 1 µl 10 µl 20 µl
1 - 6.5 µl 2 µl 0.5 µl 1 µl 10 µl 20 µl
  • Incubate the Gibson Assembly for 1 h at 50°C in Thermocycler

Electroporation

In the next step, we purify 10 µl with Isoprop purification protocol and another 5 µl we dilute with ddH2O (10 µl)

Sample Amount Used in Elektroporation
0 A 5 µl + 10 µl ddH2O
  • 1 µl
  • 14 µl
0 B 20 µl isoprop purified
  • 20 µl
1 A 5 µl + 10 µl ddH2O
  • 1 µl
  • 14 µl
1 B 20 µl isoprop purified
  • 20 µl


Colony-PCR CP.W18.A

  • 10 colonies of plate 1-8 (names are explained in the following table)
  • PC = picked colony
  • S5 = Sample 5 (watch names above on construct DelH-BB)
Template 10 x 1 PC S5 50 x 1 PC S5 4 x 1 PC S3 4 x 1 PC S3
Expected length [bp] 663 663 663 663
Named A -J (5 colonies per tube) A -J (5 colonies per tube) 1-4 1-4
Primer fw 10 µM 10 x 1 µl VF2 10 x 1 µl VF2 4 x 1 µl VF2 4 x 1 µl VF2
Primer rev 10 µM 10 x 1 µl DN07 10 x 1 1 µl DN07 1 µl DN07 1 1 µl DN07
Dream-Taq Polymerase (2x) 10 x 10 µl 10 x 10 µl 4 x 10 µl 4 x 10 µl
ddH2O 10 x 8 µl 10 x 8 µl 4 x 8 µl 4 x 8 µl
Cycles Temperature Screening start [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
14 95 60
65 30
72 45
1 72 5 min
1 12 inf

Result

Expected band: 663 bp

Fig.18.3 colony PCR of Gibson assembled DelH-BB without mRFP & tetR (loaded 7 µL of PCR) with DN07 & VF2
l1: 2log ladder, l2-5: colony PCRs from Del-rest Team, l6-13: colony PCRs from colonies 32,33,35,39,41,42,46
there is no specific bright band at the expected band of 663 bp
Fig.18.2 colony PCR of Gibson assembled DelH-BB without mRFP & tetR (loaded 7 µL of PCR) with DN07 & VF2
l1: 2log ladder, l27-50: colony PCRs from colonies 26-50
colony 32,33,35,39,41,42,46 shows a shadow specific band at ~600 bp, we expect 663 bp
Fig.18.1 colony PCR of Gibson assembled DelH-BB without mRFP & tetR (loaded 7 µL of PCR) with DN07 & VF2
l1: 2log ladder, l2-26: colony PCRs from colonies 1-25
l16colony 15 shows a shadow specific band at ~600 bp, we expect 663 bp


Colonies 15, 32, 33, 35, 39, 41, 42 and 46 show a shadow specific band at ~600 bp, slightly too low. =>  ?????

Amplification of DelH G0

Gradient-PCR Conditions G0.W18.A

Reagent G0 G0 G0 G0
Expected length [Kb] 18 18 18 18
Named G0 65 G0 66 G0 67 G0 68
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 1 µl DN11 1 µl DN11 1 µl DN11 1 µl DN11
Primer rev 10 µM 1 µl HM08 1 µl HM08 1 µl HM08 1 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl
DMSO 1 1 1 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65/66/67/68 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.18.5 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G0 amplified at 65°C, l3:G0 amplified at 66°C, l4:G0 amplified at 67°C, l5: G0 amplified at 68°C,
l4: G0 at 67°C was cut out
Fig.18.4 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G0 amplified at 65°C, l3:G0 amplified at 66°C, l4:G0 amplified at 67°C, l5: G0 amplified at 68°C,
l2-5: show slightly band at expected 18 Kb, the 5 Kb band is observed at every temperature, the best temperature seems to be 67°C

Gel shows unspecific band at 5 Kb. G0 was best amplified at 67°C.

=> Fragment was cut and gel isolated.


Re-PCR Conditions G0.W18.B

Reagent G0 G0
Expected length [Kb] 18 18
Named G0 G0 1
Template 1 µl gel extracted G0 from 22-08 1 µl gel extracted G0 from 26-08
Primer fw 10 µM 1 µl DN11 1 µl DN11
Primer rev 10 µM 1 µl HM08 1 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 6 µl 6 µl
DMSO 1 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.18.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G0 , l3: G0 , l4: G1/2a
a slight band can be observed at 18 Kb, but 5 Kb is much more amplified

A slight band can be observed at 18 Kb, but 5Kb is much more amplified.

=> By NCBI-Blast we found out that primer HM08 can also bind inside of DelH with 6 mismatches and the forward primer DN11 can also bind with 4 mismatches inside the DelH = possible explanation for 5 Kb.
=> Change the amplification conditions.


PCR Conditions G0.W18.C

Reagent G0 G0 G0
Expected length [Kb] 18 18 18
Named 0 1 2
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 1 µl DN11 1 µl DN11 1 µl DN11
Primer rev 10 µM 1 µl HM08 1 µl HM08 1 µl HM08
Phusion II Polymerase (Hot Start) 0.2 µl 0.2 µl 0.2 µl
dNTPs 0.4 µl 0.4 µl 0.4 µl
GC buffer (5x) 4 µl 4 µl 4 µl
ddH2O 12.4 µl 11.4 µl 10.4 µl
DMSO 0 µl 1 µl 2 µl
Cycles Temperature [°C] Time [s]
1 98 30
30 98 10
65 20
72 5:00 min
1 72 10 min
1 12 inf

Result

Expected band: 18 Kb

Fig.18.7 gel of amplified DelH-fragment with different DMSO concentrations and a GC-buffer (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G0 without DMSO , l3: G0 with 1 µl DMSO, l4:G0 with 2 µl DMSO,
only l4 with 2 µl DMSO shows any band, but not the expected at 18 Kb, only the band at 5 Kb.

Only PCR with 2 µl DMSO shows any band, but not the expected at 18 Kb, only the band at 5 Kb.

=> Further improve PCR condtions.


Amplification of DelH G1/2a

Re-PCR Conditions G1/2a.W18.A

Reagent G1/2a
Expected length [Kb] 18
Named G1/2a
Template 1 µl gel extracted G0 from 22-08
Primer fw 10 µM 1 µl DN11
Primer rev 10 µM 1 µl HM06
Phusion Flash Ready Mix 10 µl
ddH2O 6 µl
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.18.6 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G0 , l3: G0 , l4: G1/2a
a slight band can be observed at 18 Kb, but 5 Kb is much more amplified

Expected fragment was amplified, but gel shows numerous side bands.

=> Fragment was cut and gel isolated.


Gradient-PCR Conditions G1/2a.W18.A

Reagent G1/2a G1/2a G1/2a G1/2a
Expected length [Kb] 13 13 13 13
Named 1 2 3 F
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl gel extracted G1/2a fragment
Primer fw 10 µM 1 µl DN11 1 µl DN11 1 µl DN11 1 µl DN11
Primer rev 10 µM 1 µl HM06 1 µl HM06 1 µl HM06 1 µl HM06
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 5 µl 4 µl 6 µl
DMSO 1 2 3 1
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
67 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Fig.18.7 gel of amplified DelH-fragment (loaded 20 µL of PCR)
l1: 1Kb+ ladder, l2: G1/2a with 1 µl DMSO, l3:G1/2a with 2 µl DMSO, l4:G1/2a with 3 µl DMSO, l5: G1/2a with 1 µl DMSO amplified from the fragment itself
no band at 18 Kb
=> There is no band at 18 Kb visible - entire DelH fragment could not be amplified.


Amplification of DelH FS64 - FS71

PCR Conditions FS64-FS71.W18.A

Reagent DN11-FS66 FS67-FS68 FS69-HM08 FS69-FS70 FS71-HM08 FS67 -HM08
Expected length [Kb] 4.7 4.0 9.7 7.0 2.8 14.0
Named 1 2 3 4 5 6
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl DN11 2.5 µl FS67 2.5 µl FS69 2.5 µl FS69 2.5 µl FS71 2.5 µl FS67
Primer rev 10 µM 2.5 µl FS66 2.5 µl FS68 2.5 µl HM08 2.5 µl FS70 2.5 µl HM08 2.5 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 3 µl 3 µl 3 µl 3 µl 3 µl 3 µl
DMSO 1 1 1 1 1 1
Cycles Temperature DN11-FS66 [°C] Time [s]
1 98 5
12 98 1
68↓ 5
72 1:25 min
18 98 1
66 5
72 85
1 72 6 min
1 10 inf
Cycles Temperature [°C] Time FS67 - FS68 Time FS69-HM08 Time FS69-FS70 FS67 -HM08 Time FS71-HM08
1 98 5 5 5 5 5
12 98 1 1 1 1 1
66↓ 5 5 5 5 5
72 70 3:00 min 2:10 min 4:38 min 52
18 98 1 1 1 1 1
64 5 5 5 5 5
72 70 3:00 min 2:10 min 4:38 min 52
1 72 5 min 10 min 5 min 5 min 5 min
1 10 inf inf inf inf inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected bands: DN11-FS66 = 4.7 Kb, FS67-FS68 = 4.0 Kb, FS69-HM08 = 9.7 Kb, FS69-FS70 = 7.0 Kb, FS71-HM08 = 2.7 Kb, FS67-HM08 = 14 Kb

Fig.18.8 Gel of amplified DelH-fragments(with different new primer combinations) (loaded 20 µL)
l1:2log ladder, l2: DelH amplified with DN11 & FS66, l3:DelH amplified with FS67 & FS68, l4:DelH amplified with FS71 & HM08,
l2: shows expected band at 4.7 Kb, 4.0 Kb and 2.7 Kb => all 3 bands were cut.

Gel shows expected fragments of DN11-FS66, FS67-FS68 and FS71-HM08

=> Bands were cut. Run gel with remaining sample for gel extraction.
=> Optimize remaining PCRs.


Amplification of DelH DN11-FS66

PCR Conditions DN11-FS66.W18.A

Reagent 4x DN11 - FS66
Expected length [Kb] 4.7
Named 1
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl DN11
Primer rev 10 µM 2.5 µl FS66
Phusion Flash Ready Mix 10 µl
ddH2O 3 µl
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
68↓ 5
72 85
18 98 1
66 5
72 85
1 72 6 min
1 10 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 4.7 Kb

Fig.18.9 Gel of amplified DelH-fragments(with different new primer combinations) (loaded 20 µL)
l1:2log ladder, l2-5: DelH amplified with DN11 & FS66, l6-9:DelH amplified with FS67 & FS68, l10:DelH amplified with FS67 & HM08, l11:DelH amplified with FS71 & HM08
l2-5: shows expected band at 4.7 Kb, l6-9: at 4.0 Kb, l10 doesn't show band at 14 Kb, l11:show band at 2.7 => all specific bands were cut out

Bands could not be used, because too much UV light was applied.

Amplification of DelH FS67-FS68

PCR Conditions FS67-FS68.W18.A

Reagent 4x FS67-FS68
Expected length [Kb] 4.0
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl FS67
Primer rev 10 µm 2.5 µl FS68
Phusion Flash Ready Mix 10 µl
ddH2O 3 µl
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66↓ 5
72 1:10 min
18 98 1
64 5
72 70
1 72 5 min
1 10 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 4.0 Kb

Fig.18.10 Gel of amplified DelH-fragments(with different new primer combinations) (loaded 20 µL)
l1:2log ladder, l2-5: DelH amplified with DN11 & FS66, l6-9:DelH amplified with FS67 & FS68, l10:DelH amplified with FS67 & HM08, l11:DelH amplified with FS71 & HM08
l2-5: shows expected band at 4.7 Kb, l6-9: at 4.0 Kb, l10 doesn't show band at 14 Kb, l11:show band at 2.7 => all specific bands were cut out

Bands could not be used, because too much UV light was applied.

Amplification of DelH FS71-HM08

PCR Conditions FS71-HM08.W18.A

Reagent FS71-HM08
Expected length [Kb] 2.8
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl FS71
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 10 µl
ddH2O 3 µl
DMSO 1
Cycles Temperature A [°C] Time Temperature B [°C] Temperature C [°C]
1 98 5 98 98
12 98 1 98 98
68↓ 5 - 69
72 52 72 72
18 98 1 98 98
66 5 - 69
72 52 72 72
1 72 6 min 72 72
1 10 inf 10 10
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 2.8 Kb

Fig.18.11 Gel of amplified DelH-fragments(with different new primer combinations) (loaded 20 µL)
l1:2log ladder, l2-5: DelH amplified with DN11 & FS66, l6-9:DelH amplified with FS67 & FS68, l10:DelH amplified with FS67 & HM08, l11:DelH amplified with FS71 & HM08
l2-5: shows expected band at 4.7 Kb, l6-9: at 4.0 Kb, l10 doesn't show band at 14 Kb, l11:show band at 2.7 => all specific bands were cut out

Bands could not be used, because too much UV light was applied.

PCR Conditions FS71-HM08.W18.B

Reagent FS71-HM08
Expected length [Kb] 2.8
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl FS71
Primer rev 10 µM 2.5 µl HM08
Phusion Flash Ready Mix 10 µl
ddH2O 3 µl
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
72.3 5
72 52
30 98 1
66 5
72 52
1 72 6 min
1 10 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 2.8 Kb

Fig.18.12 Gel of amplified DelH-fragments(with different FS71 & HM08) (loaded 20 µL)
l1:2log ladder, l2: used constant 68°C annealing, l3:used touch down PCR with 70°C annealing
l2 and l3 show specific band = was cut

Both condistions resulted in the expected fragment.

=> Fragments were cut and gel extracted.
=> Run new PCR with 72.3°C constant annealing temperature because of the slight side bands.


Amplification of DelH FS69-FS70

PCR Conditions FS69-FS70.W18.A

Reagent FS69-FS70
Expected length [Kb] 7.0
Named FS69-FS70
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl FS69
Primer rev 10 µM 2.5 µl FS70
Phusion Flash Ready Mix 10 µl
ddH2O 3 µl
DMSO 1
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66↓ 5
72 2:10 min
18 98 1
64 5
72 2:10 min
1 72 6 min
1 10 inf
  • Lid preheated at 98°C
  • No hot start

Characterization of Amplified DelH Fragments

Test Restriction Digest

Fragment Fragment length [Kb] Digestes with following enzymes Expected bands [Kb]
DN11-FS66 4.7 EcoRV 3.7 & 0.9
FS67-FS68 4.0 NotI 2.7 & 1.3
FS69-FS70 7.0 SalI 5.5 & 1.6
FS71-HM08 2.8 NotI & HindIII 1.3 & 1.1 & 0.3
Fragment Amount in digest Enzyme Buffer ddH2O Final amount
DN11-FS66 5 µl 1 µl EcoRV 2 µl 3.1 buffer 10.9 µl 20 µl
FS67-FS68 5 µl 1 µl NotI 2 µl 3.1 buffer 10.9 µl 20 µl
FS69-FS70 5 µl 1 µl SalI-HF 2 µl CutSmart buffer 12 µl 20 µl
FS71-HM08 5 µl 1 µl NotI-HF & HindIII-HF 2 µl CutSmart buffer 11 µl 20 µl

Result

Expected banda are listed above.

Fig.18.14 Test restriction digest
l1:2log ladder, l2: DN11-FS66 (EcoRV), l3:FS67-FS68 (NotI) l4:FS69-FS70 (SalI), l5:FS71-HM08 (NotI & HindIII)

The test digest showed that the first fragment is cut, because the band is around 3.7 Kb, but the third and fourth lane on the gel picture show the complete non-digested fragment and on line 5 the FS71-HM08 fragment is not visible.

=> check again the digests.


Generation of DelH Plasmid pHM04 and pHM05 30-08

Gibson Assembly

Fragment Concentration [ng/µl] Amount with BB16 & tetR [µl] Amount with BB17 [µl] Amount with mRFP BB of pHM03
DN11-FS66 172.5 1.67 1.47 1.67
FS67-FS68 120.7 2.03 1.79 2.03
FS69-FS70 211.3 2.03 1.79 2.03
FS71-HM08 146 1.18 1.04 1.18
BB16 40.3 2.54 0 0
tetR (1:10) 13.2 0.56 0 0
BB17 23.0 0 3.91 0
BB+mRFP (pHM03) 0 0 0 3.08

Electroporation of pHM04

10 µl H2O were added to 5 µl of the Gibson assembly mix. 1 µl and 14 µl were electroporated into electrocompetent DH10ß. Cells were incubated at 37°C for 1 h and streaked (10% vs. 90%) on LB Amp plates and stored ON at 37°C.

Colony-PCR Conditions CP.W18.B

14 colonies (#7 was left out) were picked and screened for correct integration of first DelH fragmenta s well as grown in 500 µl LB Amp.

Template DelH construct pHM04 30-08
Expected length [bp] 663
Named Colonies 1 - 15
Primer 10 µM fw 2 µl VF2
Primer 10 µM rev 2 µl DN07
Dream-Taq Polymerase (2x) 10 µl
ddH2O 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
66 (touchdown -0.5°C) 30
72 2:30 min
18 95 60
65 30
72 2:30 min
1 72 5 min
1 12 inf

Result

Expected band: 663 bp

Fig.18.15 Colony PCR for sceening if first DelH fragment was integrated in backbone.
l1:1Kb plus ladder, l2-l7: colonies 1-6, l8-l12: colonies 8-12, l13-l14: 1Kb plus ladder, l15-l7: colonies 13-15

Colonies 1, 3, 4, 6, 12 and 15 show positive band.

=>Of the 250 µl liquid culture, screening PCRs for the correct insertion of the last DelH fragment were performed. To the remaining 250 µl, 8 ml LB Amp were added. Colonies 1 and 3 did not grow and were excluded from further analysis.


Colony-PCR CP.W18.C

Template pHM04 30-08
Expected length [Kb] 2.5
Named Colonies 1 - 15
Primer 10 µM fw 2 µl HM13
Primer 10 µM rev 2 µl VR
Dream-Taq Polymerase (2x) 10 µl
ddH2O 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
65 (touchdown -0.5°C) 30
72 2:30 min
18 95 60
65 30
72 2:30 min
1 12 inf

Results

Expected band: 2.5 Kb

Fig.18.16 Colony PCR for sceening if last DelH fragment was integrated in backbone.
l1:2log ladder, l2: colony 6, l3: colony 12, l4: colony 4, l5: colony 15

Colonies 4, 6, 12 and 15 show positive band. => Of 2 ml of the cultures, glycerol stocks were prepared. 3 ml were mini preped and test digested. The remaining 3 ml were stored.

Test Restriction Digest of Colony S4

Clone Concentration [ng/µl] Amount in digest Enzyme Buffer ddH2O Final amount
pHM04 S4 400 ng/µL 1 µl 1 µl EcoRV 1 µl CutSmart buffer 8 µl 10 µl
pHM04 S4 400 ng/µL 1 µl 1 µl NotI 1 µl CutSmart buffer 8 µl 10 µl
pHM04 S4 400 ng/µL 1 µl 1 µl SalI-HF 1 µl CutSmart buffer 8 µl 10 µl

Result

Expected bands are shown in table below.

Clone Clone length [bp] Digestes with following enzymes Expected bands [bp]
pHM04 S4 22,937 EcoRI 17,829 & 5,108
pHM04 S4 22,937 NotI 11,079, 6201, 3,998 & 1,628
pHM04 S4 22,937 PvuI 11,621, 8628 & 2,685
Fig.18.17 Test restriction digest of pHM04
l1:2log ladder, l2: pHM04 (EcoRI), l3:pHM04 (NotI) l4:pHM04 (PvuI)
The expected bands were not there. So the colony seems not to have the entire DelH-plasmid. .