Team:Heidelberg/Templates/Del week16 AF

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Contents

12-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step; run at 135 V, 0.8 % gel (TAE)(11.08)
Amplification of DelAF using gradient PCR, Amplification of DelOP 72°C 2-step after cutting; run at 135 V, 0.8 % gel (TAE)(11.08)
Reaction
Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
67.5 - 65.0 (ΔT = 0.5) ↓ 0.5 5
72 3:20
18 98 1
65.5 - 63.0 (ΔT = 0.5) 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked well, gradient displays an optimal annealing temperature of 65.5°C, which will be used for further amplifications
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

13-08-2013

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 12-08-2013 with EcoRI-HF

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (12-08-2013) 20
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • Weak bands of about 4.6kbp visible, as well as on of about 2.6kbp
  • digest will be repeated using higher concentrations of DNA to clearify results of restriction digest

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (FS_02-FS_05); run at 100 V, 0.8 % gel (TAE)(13.08)
Amplification of DelAF (FS_02-FS_05), cut; run at 100 V, 0.8 % gel (TAE)(13.08)
Reaction

1 sample contains 2µL of DMSO

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 4.5 µL FS_02
Primer rev 4.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked though a slight smear occured, therefore PCR will be repeated on the more precise Biometra TProfessional Basic cylcer again
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit

14-08-2013

Concentration measurement (FS_02 to FS_05; 11.2 kb)

Fragment Primer Date PCR Concentration
DelAF FS02-FS05 11-08-2013 ~10 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL
DelAF FS02-FS05 12-08-2013 0 ng/µL

Results:

  • concentrations are not sufficient for gibson assembly
  • PCR will be repeated and gel slices of different reactions will be pooled for one gel extraction using QIAquick Gel Extraction Kit to obtain the concentrations needed for gibson assembly

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 11-08-2013 with EcoRI-HF

Test restriction digest of fragment FS_02-FS_05 (12-08) with EcoRI; run at 100 V, 0.8 % gel (TAE)(13.08)


Incubation at 37°C for 2 hours 45min

what µL
FS_02 to FS_05 (11-08-2013) 18
EcoRI-HF 1
CutSmart Buffer 2.5
dd H2O 3.5

Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp

Results:

  • One band of about 5kbp and one of 2.5kbp
  • no clear result, digest will be repeated with another enzyme (ClaI), as the enzyme used was beyond expiration date

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 10-08-2013 with ClaI

Test restriction digest of fragment FS_02-FS_05 (12-08) with ClaI; run at 100 V, 0.8 % gel (TAE)(14.08)

Incubation at 37°C for 2 hours

what µL
FS_02 to FS_05 (10-08-2013) 20
ClaI 1
CutSmart Buffer 2.5
dd H2O 1.5

Expected fragment sizes: 6.9kbp, 4.3kbp

Results:

  • restriction digest displays the expected fragments, therefore amplification of the desired fragment can be assumed
  • PCR product will be prepared for sequencing by GATC to proof amplification of the desired DNA sequence before Gibson Assembly

17-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (17-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (17-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
Phusion Ready Mix 10 µL
DMSO 1 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out, pooled and DNA purified using QIAquick Gel Extraction Kit to obtain concentrations needed for gibson assembly

18-08-2013

Amplification from FS_02 to FS_05; 11.2 kb

Amplification of DelAF (18-08); run at 100 V, 0.8 % gel (TAE)
Amplification of DelAF, cut (18-08); run at 100 V, 0.8 % gel (TAE)
Reaction of DelAF

6x20 µL

Reagent DelAF
Template D.acidovorans SPH-1 colony
Primer fw 2.5 µL FS_02
Primer rev 2.5 µL FS_05
DMSO 1 µL
Phusion Ready Mix 10 µL
dd H2O 4 µL
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65.5 ↓ 0.5 5
72 3:20
18 98 1
63.5 5
72 3:20
1 72 10min
1 10 inf

Results:

  • Amplification of DelAF worked
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit
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