Difference between revisions of "Colony PCR"

Revision as of 15:42, 21 October 2009

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