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Team:Heidelberg/Templates/Del week14 AG
From 2013.igem.org
30-07-2013
Amplification from FS_02 to FS_26; 16.7 kb
File:20130730 2log FS2-26.200nMor400nM65°Cconst FS2-26.200or400nM.68°Ctd 2xFS02-05200nM65const 2xFS02-05400nM65const.png
Amplification of Del A-G under different conditions and DelAF; run at 100 V 0.8% agarose gel (TAE)
4 reactions, 68°C Touchdown and 65°C constant with 200nM and 400nM Primers
- Reaction
| what | µl |
|---|---|
| D. acidovorans | 1 |
| FS_02: (1/10) | 2/4 |
| FS_26: (1/10) | 2/4 |
| 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 | 5:40 | |
| 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 | 5:40 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 5:40 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAG did not work, neither annealing at 65°C constant nor a touchdown starting from 68°C led to the desired product
