Team:Paris Saclay/Protocols/PCR for bacterial culture
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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 | 10X | 1X | 5μl |
dNTPs | 10mM | 200μM | 1μl |
Primer A | 100μM | 0.5μM | 2.5μl |
Primer B | 100μM | 0.5μM | 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.