Making long term stocks | Transformation | [http://microscopy.berkeley.edu/Resources/instruction/buffers.html Buffer recipes] | Colony PCR

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Colony PCR

Materials

• 2x Phusion Mastermic
• Primers
• Nuclease free H2O
• Cell culture!

Method

• Pick a single colony and place in 20µL of H20.
• Cell lysis:
  • Incubate 10 min @ 98°C
  • Freeze 10 min @ -80°C
  • Vortex for 2-5 min
• In a PCR tube mix:
  • 10µL of 2X Phusion Mastermix
  • 1µL of DNA template (lysate
  • XµL of Primer 1
  • XµL of Primer 2
  • 7µL of Nuclease free H2O
• Run PCR:
  • Initial Denaturation: 30s @ 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]