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Team:EPF-Lausanne/Protocols/1.5 steps PCR
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Latest revision as of 18:44, 20 October 2009
1. Do the following mixing:
| dNTP | 1 μl |
|---|---|
| 5x Buffer + MgCl2 | 10 μl |
| Primer Mix | 1 μl |
| template | 0.5 μl |
| HiFi Plus DNAp | 0.5 μl |
| dH2O | 36.5 μl |
| Final Volume | 50 μl |
2. Make it run 10 cycles:
| 1 | 94°C | 4:00 |
|---|---|---|
| 2 | 94°C | 0:30 |
| 3 | 55°C | 1:00 |
| 4 | 72°C | 2:00 |
| 5 | Cycle 2-4 10 times | |
| 6 | 72°C | 7:00 |
| 7 | 4°C | ∞ |
3. We now add the standard iGEM primers which finish the extension, and do the following cycles:
| 1 | 94°C | 4:00 |
|---|---|---|
| 2 | 94°C | 0:30 |
| 3 | 55°C | 1:00 |
| 4 | 72°C | 2:00 |
| 5 | Cycle 2-4 25-30 times | |
| 6 | 72°C | 7:00 |
| 7 | 4°C | ∞ |
