Basic information
Date:
Mon, 2007-12-03 00:00
Gene:
Template:
dna
Procedure
PCR cocktail:
| Component | Volume (ul) | Mix (ul) |
| Forward primer | 0.5 | |
| Reversed primer | 0.5 | |
| Buffer | ||
| MgCl2 | ||
| dNTPs | ||
| Taq | ||
| DNA (if nested) | 2 | |
| ddH2O |
22 | |
| Total volume | 25 |
Forward primer(s):
Thermal cycling (abbreviated):
95ºC@4min, 2X (94ºC@30sec,59-54ºC@30sec, 72ºC@30sec),35X (94ºC@30sec,52ºC@30sec, 72ºC@90sec),72ºC@10min
PCR machine:
GeneAmp
Thermal cycling:
| Step | Comment | Cycles | Time (sec) | Temperature (°C) |
| Initialization | For DNA polymerases that need hot-start heat activation | 1 | ||
| Denaturation | DNA melting producing single-stranded DNA | 1 | 240 | 95 |
| Amplification | 2 | |||
| Denaturation | 30 | 94 | ||
| 30 | 59-55 | |||
| 30 | 72 | |||
| Amplification | 35 | |||
| Denaturation | 30 | 94 | ||
| Annealing | 30 | 52 | ||
| Extension | 90 | 72 | ||
| Final elongation | Any remaining single-stranded DNA is extended | 1 | 600 | 72 |
| Hold | 1 | 8 |
Reversed primer(s):
People:
Products and Equipment:
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