Team:Newcastle/Colony PCR
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====Conditions for ThermoCycler:==== | ====Conditions for ThermoCycler:==== | ||
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+ | # Initialise - 95°C for 2 minutes. | ||
+ | # Denature - 95°C for 30 seconds. | ||
+ | # Anneal - 52°C for 30 seconds (Melting temperature, Tm of template). | ||
+ | # Extension - 75°C for 30 seconds | ||
+ | # Extension finish - 75°C for 5 minutes | ||
+ | # Hold - 4°C | ||
+ | |||
+ | Steps 2 to 4 are repeated for 30 cycles before continuing to step 5. |
Revision as of 13:42, 26 July 2010
Colony PCR
Materials to add accordingly:
- 37.5µl of distilled H2O
- 10µl of 5x GoTaq Buffer
- Nucleotide DNTP
- 2.5µl forward primer
- 2.5µl backward primer
- 1µl template DNA
Conditions for ThermoCycler:
- Initialise - 95°C for 2 minutes.
- Denature - 95°C for 30 seconds.
- Anneal - 52°C for 30 seconds (Melting temperature, Tm of template).
- Extension - 75°C for 30 seconds
- Extension finish - 75°C for 5 minutes
- Hold - 4°C
Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.