From 2013.igem.org
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