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Team:Heidelberg/Templates/Del week13 AG
From 2013.igem.org
Contents |
27-07-2013
Amplification from FS_02 to FS_26; 16.7 kb
File:20130728 2log 1kb 2xFS02-FS23.200nMor400nM 2x17kbpSophie 2log 2xconcentrationNils.png
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 SPH-1 | 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
File:20130727 2log 1kb 2-24.200nM.68td 2-24.400nM.68td 2-23.200nM.68td 2-23.400nM.68td 2-23.200nM.65 2-23.400nM.65.png
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 | 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
File:20130728 1kbruler 4xFS02-FS23.60const.@T100.200nM T100.400nM old.200nM old.400nM red.200nM red.400nM FS02-FS24.janusA.200nM.66td FS02-FS24.T100.400nM.60const 1kb FS04-FS24.png
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 | 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
