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Team:Cambridge/Protocols/ColonyPCR
From 2010.igem.org
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==Notes== | ==Notes== | ||
| - | *For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 | + | *For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR]) from James Brown we used the following values: |
**X = 1µL | **X = 1µL | ||
**Y = 65°C | **Y = 65°C | ||
*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix] | *[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix] | ||
{{:Team:Cambridge/Templates/footer}} | {{:Team:Cambridge/Templates/footer}} | ||
Revision as of 15:07, 23 August 2010
Making long term stocks | Transformation | [http://microscopy.berkeley.edu/Resources/instruction/buffers.html 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
- For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR]) from James Brown we used the following values:
- X = 1µL
- Y = 65°C
- [http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]
