Basic information
Date:
Thu, 2008-10-30 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, 58-54ºC@30sec, 72ºC@60sec), 34x(94ºC@30sec, 52ºC@30sec, 72ºC@60sec), 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 | DNA melting producing single-stranded DNA |
- | 30 | 94 |
Annealing | The primer binds to the template | - | 30 | 58-54 |
Extension | The DNA polymerase synthesizes new DNA | - | 60 | 72 |
Amplification | 34 | |||
Denaturation | 30 | 94 | ||
Annealing | 30 | 52 | ||
Extension | 60 | 72 | ||
Final elongation | Any remaining single-stranded DNA is extended | 1 | 600 | 72 |
Final hold | Short-term storage | 1 | - | 12 |
Reversed primer(s):
File attachments:
People:
Products and Equipment:
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