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Team:Hawaii/Protocols/Colony PCR
From 2008.igem.org
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| - | Protocol dictated by Dr. Sean Callahan, Department of Microbiology, University of Hawaii at Manoa | + | Protocol dictated by [http://www2.hawaii.edu/~scallaha/SMCsite/ Dr. Sean Callahan], Department of Microbiology, University of Hawaii at Manoa |
Latest revision as of 08:10, 16 July 2008
Protocol
- Pick a colony and suspend in 200 μl LB (or other appropriate media).
- Vortex ~8 sec.
- Heat at 97C for 10 min.
- Vortex again briefly.
- Use 1 μl in PCR reaction.
PCR reaction
- Combine:
- 1 μl colony sample (above)
- 0.5 μl 10mM forward primer
- 0.5 μl 10mM reverse primer
- 3 μl nanopure water
- 5 μl Taq (we used EconoTaq Green Taq)
- Run for 30 cycles of denaturing, annealing, extension
- Initial denature @ 94C for 2 min.
- Denature @ 94C for 30 sec.
- Anneal @ 62C for 30 sec.
- Extend @ 72C for 90 sec.
- Final extension @ 72C for 10 min.
- Hold @ 4C inifinitly.
Run PCR products on a gel to verify desired DNAs.
Reference
Protocol dictated by [http://www2.hawaii.edu/~scallaha/SMCsite/ Dr. Sean Callahan], Department of Microbiology, University of Hawaii at Manoa
