Colony-PCR: Difference between revisions
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==PCR Program== | ==PCR Program== | ||
* 95°C 5 min | * 95°C 5 min | ||
35 times the following cycle | |||
** 95°C 1 min | ** 95°C 1 min | ||
** 55°C 30 sek. (°C depends on your primers) | ** 55°C 30 sek. (°C depends on your primers) | ||
Revision as of 09:55, 29 August 2013
Instead of plasmid or genomic DNA, you can use a colony from the agar plate as template DNA. You should use a sterile toothpick, pick into the colony and spread it in the PCR tube.
For a 10 µl reaction (Taq Polymerase Peqlab):
- 1 µl Buffer S
- 0,2 µl dNTP's (10 mM)
- 0,2 µl Primer fw
- 0,2 µl Primer rev
- 0,04 µl Taq Polymerase
- 20,9 µl Water
PCR Program
- 95°C 5 min
35 times the following cycle
- 95°C 1 min
- 55°C 30 sek. (°C depends on your primers)
- 72°C 1 min/kb
cycle end
- 72°C 10 min
- 10°C hold
