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Team:Paris Saclay/Protocols/PCR for bacterial culture
From 2014.igem.org
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Revision as of 12:00, 29 July 2014
PCR for bacterial culture
- 1. In a cold PCR tube (Eppendorf), pipet in the presented order the solutions listed in Table 1.
| Component | Initial concentration | Final concentration | Example for 50μl |
|---|---|---|---|
|
H2O |
- |
Add to final volume |
27.75μl (50μl final) |
| Tp Green 5X | 5X | 1X | 10μl |
| dNTPs | 10mM | 200μM | 1μl |
| Primer A | 1mM | 10μM | 2μl |
| Primer B | 1mM | 10μM | 2μl |
| Culture | - | - | 2μl |
| GoTaq DNA polymerasse | - | - | 0.25μl |
| DMSO | - | - | 1.5μl |
- 2. Program the PCR machine according to the Table 2.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
95 - 98°C |
30 s - 3 min |
1 |
| Denaturation | 95 - 98°C | 10 - 30 s | 25 - 30 |
| Annealing | variable | 30 s | 25 - 30 |
| Extension | 72°C | 30 s - 2 min | 25-30 |
| Final extension | 72°C | 10 min | 1 |
| Final extension | 4 - 8°C | hold | 1 |
- 3. Verify correct amplification by agarose gel electrophoresis.
