From 2013.igem.org
27-07-2013
Amplification from FS_02 to FS_26; 16.7 kb
2 x Amplification of FS02 to FS_23 27.07.13 200nM/400nM, 4x 2 x Amplification of FS02 to FS_26 27.07.13; run at 100V
- Reaction
| what | µl
|
| D. acidovorans DSM-39 | 1
|
| FS_02: (1/10) | 2
|
| FS_26: (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
|
| 58 | 5
|
| 72 | 3:50
|
| 1 | 72 | 12 min
|
| 1 | 10 | inf
|
Results:
- Amplification of DelAG did not work, unspecific band at approximately 3.5 kbp occured
Amplification 1 from FS_02 to FS_23; 22.8 kb
2 x Amplification of FS02 to FS_24 27.07.13 68°C Touchdown 200nM/400nM; 4x Amplification of FS-02 to FS23 27.0713 1 & 2 68°C Touchdown 200nM/400nM; 3. & 4. 65°C const. 200/400 nM, run at 100V
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
- Reaction
| what | µl
|
| D. acidovorans DSM-39 | 1
|
| FS_02: (1/10) | 4/2
|
| FS_23: (1/10) | 4/2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 0/4
|
- Conditions I
| Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 65 | 5
|
| 72 | 6:40
|
| 1 | 72 | 12 min
|
| 1 | 10 | inf
|
- Conditions II
| Biometra TProfessional Basic
|
| Cycles-PCR | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 12 | 98 | 1
|
| 68 ↓ 0.5 | 5
|
| 72 | 6:40 min
|
| 18 | 98 | 1
|
| 66 | 5
|
| 72 | 6:40 min
|
| 1 | 72 | 10 min
|
| 1 | 4 | inf
|
Results:
- Amplification of DelAG did not work, several unspecific bands occured
Amplification 2 from FS_02 to FS_23; 22.8 kb
1kbruler / 4xFS02-FS23 60const @T100 200nM; T100 400nM; old 200nM; old 400nM ; red 200nM; red 400nM / FS02-FS24 66td janusA 200nM / FS02-FS24 T100 60const 200nM; 400nM 1kb FS04-FS24.png ; run at 100 V, 0.8 % gel (TAE)
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
- Reaction
| what | µl
|
| D. acidovorans DSM-39 | 1
|
| FS_02: (1/10) | 4/2
|
| FS_23: (1/10) | 4/2
|
| Phusion flash Master Mix | 10
|
| DMSO | 1
|
| dd H2O | 0/4
|
- Conditions I
| Biorad MyCycler, Biometra TProfessional Basic and Biorad T100
|
| Cycles | temperature [°C] | Time [s]
|
| 1 | 98 | 10
|
| 30 | 98 | 1
|
| 60 | 5
|
| 72 | 6:40
|
| 1 | 72 | 12 min
|
| 1 | 10 | inf
|
Results:
- Amplification of DelAG did not work, independent of the cycler only unspecific bands at 4.0 and 5.2 kbp occured
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