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Team:Heidelberg/Templates/Del week14 OP
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Revision as of 01:44, 30 September 2013
Contents |
01-08-2013
Amplification from FS_22 to FS_13(s); 2.7 kb
- Reaction
| what | µl |
|---|---|
| D.acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short/FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 92 | 5 | |
| 91 | 5 | |
| 90 | 5 | |
| 89 | 5 | |
| 88 | 5 | |
| 87 | 5 | |
| 86 | 5 | |
| 85 | 5 | |
| 84 | 5 | |
| 82 | 5 | |
| 80 | 5 | |
| 78 | 5 | |
| 76 | 5 | |
| 74 | 5 | |
| 72 | 5 | |
| 70 | 5 | |
| 68 | 5 | |
| 66 | 5 | |
| 64 | 5 | |
| 62 | 5 | |
| 60 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification of DelOP did not work with stepwise cooling to the intended annealing temperature of 60°C
07-08-2013
Amplification from FS_22 to FS_13; 2.7 kb
_3x14-15(L)besch.png)
_3x14-15(L)cut.png)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_22: (1/10) | 2 |
| FS_13: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Amplification from FS_22 to FS_13; 2.7 kb
_DelH_1kbbesch.png)
_DelH_1kb_cut.png)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_22: (1/10) | 2 |
| FS_13: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP led to a band of the desired size as well as a smear and several different sideproducts
- Band was cut out and DNA purified using QIAquick Gel Extraction Kit to be restriction digested for validation of the PCR product
- Amplification will be repeated at lower temperature to obtain more product
