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Team:Paris Saclay/Protocols/PCR for bacterial culture
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|- | |- | ||
| Tp Green 5X | | Tp Green 5X | ||
| - | | | + | | 5X |
| 1X | | 1X | ||
| 5μl | | 5μl | ||
| Line 31: | Line 31: | ||
|- | |- | ||
| Primer A | | Primer A | ||
| - | | | + | | 1mM |
| - | | | + | | 10μM |
| 2.5μl | | 2.5μl | ||
|- | |- | ||
| Primer B | | Primer B | ||
| - | | | + | | 1mM |
| - | | | + | | 10μM |
| 2.5μl | | 2.5μl | ||
|- | |- | ||
Revision as of 11:51, 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 |
37.75μl (50μl final) |
| Tp Green 5X | 5X | 1X | 5μl |
| dNTPs | 10mM | 200μM | 1μl |
| Primer A | 1mM | 10μM | 2.5μl |
| Primer B | 1mM | 10μM | 2.5μl |
| Template DNA: Genomic DNA | - | 1μl | 1μl |
| GoTaq DNA polymerasse | - | 0.25μl | 0.25μl |
- 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.
