Team:Cambridge/Protocols/ColonyPCR
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(New page: {{:Team:Cambridge/Templates/header}} {{:Team:Cambridge/Protocol Menu}} =Colony PCR= ==Materials== *2x Phusion Mastermic *Primers *Nuclease free H2O *Cell culture! ==Method== *Pick a sing...)
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(New page: {{:Team:Cambridge/Templates/header}} {{:Team:Cambridge/Protocol Menu}} =Colony PCR= ==Materials== *2x Phusion Mastermic *Primers *Nuclease free H2O *Cell culture! ==Method== *Pick a sing...)
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Revision as of 15:04, 23 August 2010
Making long term stocks | Transformation | Buffer recipes | Colony PCR
Colony PCR
Materials
- 2x Phusion Mastermic
- Primers
- Nuclease free H2O
- Cell culture!
Method
- Pick a single colony and place in 20µL of H20.
- In a PCR tube mix:
- 10µL of 2X Phusion Mastermix
- 1µL of DNA template
- XµL of Primer 1
- XµL of Primer 2
- 7µL of Nuclease free H2O
- Run PCR:
- Initial Denaturation: 15 min @ 98°C
- Denaturation: 10s @ 98°C
- Annealing: 30s @ Y°C
- Elongation: 30s per kb @ 72°C
- Reapeat steps 2-4, 25 to 35 times
- Final Elongation: 10 min @ 72°C
- Final Hold: ∞ @ 4°C
Notes