Team:Heidelberg/Templates/Del week13 EG

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Contents

24-07-2013

Restriction digest of fragment FS_04 to FS_07; 11.1 kb with PvuI-HF

Test restriction digest of DelEG FS04 to FS07 (24.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 45 min

what µl
FS_04 to FS_07 (14-07-2013 and 15-07-2013) 15
PvuI-HF 0.8
Buffer CutSmart 2
dd H2O 2.8
Expected fragment lengths [bp] 6187, 4917

Results:

  • restriction digest did not work
  • digest will be repeated with newly amplified and purified DelEG


28-07-2013

Amplification from FS_04 to FS_09 ; 14.4 kb

2 reactions with conditions I and II

Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 4:40
1 72 13 min
1 10 inf
Conditions II
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 4:40
18 98 1
66 5
72 4:40
1 72 13 min
1 10 inf

Amplification from FS_26 to FS_07

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)

This amplification did not make sense, two reverse Primer were used. We mixed up Primer FS_24 with Primer FS_26.

2 reactions with conditions I and II

Reaction
what µl
D. acidovorans DSM-39 1
FS_26: (1/10) 2
FS_07: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 3:20
1 72 13 min
1 10 inf
Conditions II
Biorad C1000 Touch Block A
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 12 min
1 10 inf

Results:

  • Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
  • later on it was discovered, that primers had been mixed up

Amplification from FS_26 to FS_24

This amplification did not make sense, two reverse Primer were used.

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)

2 reactions with conditions I and II

Reaction
what µl
D. acidovorans DSM-39 1
FS_26: (1/10) 2
FS_24: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 3:20
1 72 13 min
1 10 inf
Conditions II
Biorad C1000 Block A
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3:20
18 98 1
66 5
72 3:20
1 72 12 min
1 10 inf

Results:

  • Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
  • later on it was discovered, that primers had been mixed up
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