Basic information
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, 40x(94ºC@30sec, 52ºC@30sec, 72ºC@30sec), 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 | 40 | - | - | |
Denaturation | DNA melting producing single-stranded DNA |
- | 30 | 94 |
Annealing | The primer binds to the template | - | 30 | 52 |
Extension | The DNA polymerase synthesizes new DNA | - | 30 | 72 |
Final elongation | Any remaining single-stranded DNA is extended | 1 | 600 | 72 |
Final hold | Short-term storage | 1 | - | 12 |
PCR program:
S30
Reversed primer(s):
File attachments:
People:
Products and Equipment:
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