Colony PCR

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Reagents

  • Primers
  • dNTPs (2 mM of each dNTP)
  • Expand polymerase (Roche)
  • Primers
  • Yeast Colonies

Protocol

  1. Pick about 20% of a colony and mix in 20 uL of sterile water.
  2. Prepare PCR:
  • 5 uL Expand Polymerase Buffer
  • 5 uL dNTPs
  • 1 uL of a 5X diluted stock
  • 1 uL of a 5X diluted stock
  • 5 uL cell suspension
  • 0.5 uL Expand Polymerase

Run PCR Program Colony PCR:

  1. 94C for 5 min
  2. 94C for 10s
  3. 40C for 30s
  4. 68C for 2 min
  5. Repeat Steps 2-4 10 times
  6. 94C for 10s
  7. 40C for 30s
  8. 68C for 1 min + 20s/cycle
  9. Repeat Steps 6-8 19 times
  10. 68C for 7min
  11. 4C hold forever
  • Check PCR by Agarose Gel Electrophoresis