Team:Todai-Tokyo/Protocols/Colony PCR
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== Colony PCR Protocol == | == Colony PCR Protocol == |
Latest revision as of 16:29, 20 October 2009
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Colony PCR Protocol
- Aliquot into PCR tubes 5µl of MilliQ
- Pick single colonies with a toothpick and immerse in the above MilliQ to suspend
- Put in PCR machine set at 95ºC for 5 min.
- Add to each tube 5µl of the following solution and mix by pipetting
- 0.1µl 100µM 5’ primer
- 0.1µl 100µM 3’primer
- 0.8µl 2.5mM dNTP
- 1µl 10x standard buffer
- 2.92µl MilliQ
- 0.08µl Ex-Taq
- PCR using the following program
- 95ºC 2 min
- 95ºC 30 sec
- 52-55ºC 30 sec (Depending on the Tm of the primers)
- 72.5ºC 15 sec × (# of kb of DNA to be amplified)
- 95ºC 30 sec Repeat 2-4 29 times
- 25ºC pause