Team:Hawaii/Protocols/Colony PCR

Protocol

 * 1) Pick a colony and suspend in 200 &mu;l LB (or other appropriate media).
 * 2) Vortex ~8 sec.
 * 3) Heat at 97C for 10 min.
 * 4) Vortex again briefly.
 * 5) Use 1 &mu;l in PCR reaction.

PCR reaction
 * Combine:
 * 1 &mu;l colony sample (above)
 * 0.5 &mu;l 10mM forward primer
 * 0.5 &mu;l 10mM reverse primer
 * 3 &mu;l nanopure water
 * 5 &mu;l 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 Dr. Sean Callahan, Department of Microbiology, University of Hawaii at Manoa