Team:Heidelberg/Templates/Del week13 FG

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Contents

22-07-2013

Amplification from FS_20/FS_21 to FS_09; 8.5 kb

Re-PCR of DelOP (22-13long), Amplification of DelFG (20-9 and 21-9) (22.07); run at 100 V, 0.8 % gel (TAE)
Re-PCR of DelOP (22-13long), Amplification of DelFG (20-9 and 21-9) (22.07) cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_20/FS_21: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
65 ↓ 0.5 5
72 2:20
18 98 1
63 5
72 2:20
1 72 10 min
1 12 inf

Results:

  • Amplification of DelFG did not work
  • other primers/combinations of primers will be used
  • somehow getting annoyed by this part of D. Acidovorans

Amplification from FS_20/FS_21 to FS_11_short; 11.6 kb

Amplification of DelFG (22.07); run at 100 V, 0.8 % gel (TAE)

4x 20µl (70 touchdown, 65 touchdown)

Reaction
what µl
D. acidovorans DSM-39 1
FS_20 or FS_21: (1/10) 2
FS_11_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions I
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
70 ↓ 0.5 5
72 3:40
18 98 1
68 5
72 3:40
1 72 13min
1 12 inf
Conditions II of Del FG
Biorad T100
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
65 ↓ 0.5 5
72 3:40
18 98 1
63 5
72 3:40
1 72 13min
1 12 inf

Results:

  • Amplification of DelFG did not work neither with a touchdown PCR starting at an annealing temperature of 65°C nor 70°C

Amplification from FS_20/FS_21 to FS_23; 11.6 kb

Amplification of DelFG (22.07); run at 100 V, 0.8 % gel (TAE)

4x 20µl (70 touchdown, 65 touchdown)

Reaction
what µl
D. acidovorans DSM-39 1
FS_20 or FS_21: (1/10) 2
FS_23: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions I
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
70 ↓ 0.5 5
72 3:40
18 98 1
68 5
72 3:40
1 72 13min
1 12 inf
Conditions II
Biorad T100
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
65 ↓ 0.5 5
72 3:40
18 98 1
63 5
72 3:40
1 72 13min
1 12 inf

Results:

  • Neither the amplification with the primers FS_20 to FS_23 or FS_21 to FS_23 did work. Another primer combination has to be tried.

24-07-2013

Amplification of DelFG (FS_06 to FS_07; 5.2 kb)

PCR of FG (FS_06 to FS_07) or (FS_20 to FS_07) or (FS_21 to FS_07) as indicated; run at 100 V, 0.8 % gel (TAE)
Reaction I
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1

Amplification from FS_20 to FS_07; 5.2 kb

PCR of FG (FS_06 to FS_07) or (FS_20 to FS_07) or (FS_21 to FS_07) as indicated; run at 100 V, 0.8 % gel (TAE)
Reaction II
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1

Amplification from FS_21 to FS_07; 5.2kb

PCR of FG (FS_06 to FS_07) or (FS_20 to FS_07) or (FS_21 to FS_07) as indicated; run at 100 V, 0.8 % gel (TAE)
Reaction III
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
Conditions for reactions I - III
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:10
18 98 1
64 5
72 2:10
1 72 10min
1 12 inf

Results:

  • The amplification of FS_06 to FS_07 did not work. No bands were visible
  • The amplification of FS_21 to FS_07 led to several bands, but none of these was the intended product
  • The amplfication of FS_20 to FS_07 also led to several bands, one band at the right height was observed. Consequently the specificity of the PCR weill be increased by a higher annealing temperature

Amplification from FS_20 to FS_07; 5.2 kb

2x20µl (one with conditions I, other one with conditions II)

Reaction
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
Conditions I
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 2:10
18 98 1
68 5
72 2:10
1 72 10min
1 12 inf
Conditions II
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:10
18 98 1
66 5
72 2:10
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work, several bands occured, one of these had the size of the intended product but purity of the PCR was not sufficient for Gibson Assembly
  • nethertheless bands were cut out and DNA purified using QIAquick Gel Extraction Kit for restriction digest

Amplification from FS_20 to FS_07; 5.2 kb

Amplification of DelFG II (FS20 to FS07; 24.07),; run at 100 V, 0.8 % gel (TAE)
Amplification of DelFG II (FS20 to FS07; 24.07), ; run at 100 V, 0.8 % gel (TAE)

4x20µl

Reaction
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 2:10
18 98 1
68 5
72 2:10
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work, several bands occured, one of these had the size of the intended product but purity of the PCR was not sufficient for Gibson Assembly
  • nethertheless bands were cut out and DNA purified using QIAquick Gel Extraction Kit for restriction digest

25-07-2013

Restriction digest of DelFG (FS_20 to FS_07; 5.2 kb; 23-07-2013 and 24-07-2013) with XmaI

Restriction digest of Fragment FS_20 to FS_07 (23.07 and 24.07) with XmaI The second lane is also a digest ; Expected size of digested fragments: 0.9kbp and 4.3kbp; run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 45 min

what µl
FS_20 to FS_07 (23-07-2013 and 24-07-2013) 15
XmaI 0.8
Buffer CutSmart 2
dd H2O 2.2

Results:

  • restriction digest of DelFG did not work, only very slight bands were visible
  • digest will be repeated with higher amount of DNA and enzyme to improve analysis on the gel

26-07-2013

Amplification from FS_20 to FS_07; 5.2 kb

Reaction I
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
70 5
72 2:10
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work, several bands occured, one of these had the size of the intended product but purity of the PCR was not sufficient for Gibson Assembly
  • Annealing temperature will be increased further, to optimize primer specifity

Restriction digest of fragment from FS_20 to FS_07; 5.2 kb; 23-07-2013 and 23-07-2013) with ClaI

restriction digest of FS_20 to FS_07 (26.07.13) with ClaI and concentration measurement of DelOP (25.07.13); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 45 min

what µl
FS_20 to FS_07 (23-07-2013 and 23-07-2013) 25
ClaI 1
Buffer CutSmart 3
dd H2O 1

Results:

  • restriction digest of Del FG did not lead to the expected results
  • as no DNA was visible in the restriction digest, experiment will be repeated with a higher amount of DNA

Amplification from FS_06 to FS_07; 5.2 kb

4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07); run at 100 V, 0.8 % gel (TAE)
4 x Amplification of DelFG (FS20 to FS07; 26.07), 4 x Amplification of DelOP (FS_22 to FS13; 25.07) after cutting; run at 100 V, 0.8 % gel (TAE)

4 x 20µL

Reaction I
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 4
FS_07: (1/10) 4
Phusion flash Master Mix 10
DMSO 1
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
72 ↓ 0.5 5
72 2:10
18 98 1
70 5
72 2:10
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work, several bands occured, one of these had the size of the intended product but purity of the PCR was not sufficient for Gibson Assembly
  • nethertheless bands were cut out and DNA purified using QIAquick Gel Extraction Kit for restriction digest
  • Does anyone know, why we are constantly repeating this totally deficient PCR?

Amplification from FS_6/FS_20/FS_21 to FS_24 (PRIMER FS_24 MIXED UP!)

run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_06/FS_20/FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 1:20
1 72 7 min
1 10 inf

Results:

  • no PCR product occured since the wrong primers were used

28-07-2013

Amplification from FS_21 to FS_24; (PRIMER 24 was MIXED UP!)

2log ladder / FS21-FS24 60const / FS07-FS26 65const / FS07-FS26 68const / FS24-FS26 65const / FS24-FS26 68const; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 2:30
1 72 8 min
1 12 inf
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
60 5
72 2:30
1 72 8 min
1 12 inf

Results:

  • no PCR product occured since the wrong primers were used
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