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Reagents
- Primers
- dNTPs (2 mM of each dNTP)
- Expand polymerase (Roche)
- Primers
- Yeast Colonies
Protocol
- Pick about 20% of a colony and mix in 20 uL of sterile water.
- 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:
- 94C for 5 min
- 94C for 10s
- 40C for 30s
- 68C for 2 min
- Repeat Steps 2-4 10 times
- 94C for 10s
- 40C for 30s
- 68C for 1 min + 20s/cycle
- Repeat Steps 6-8 19 times
- 68C for 7min
- 4C hold forever
- Check PCR by Agarose Gel Electrophoresis