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Team:Washington/COLONY PCR GREEN
From 2013.igem.org
Colony PCR with Green tag
(See General PCR Protocol and PCR GoTaq)
Master mix (7ul):
1ul 10uM forward primer
1ul 10uM reverse Primer
5ul 2x Green taq (GoTaq)
Cell water (3ul) “colony water”:
Pick one colony from the plate and mix with 10ul of sterile diH2O or PBS to make 10ul of cell solution. Save for inoculating overnights to miniprep.
If screening for successful clones, the cell solution should be retained
Sterile glycerol can be added to 50% final concentration, and cell solution can be frozen at -80oC for use at a later date
*Make multiple cell waters using all screened colonies and run multiple PCRs
Reaction Mixture:
Master mix (7ul) + Cell solution (3ul) = 10ul total per tube
Use program "Colony" & change the extension time (1 min per kb)
PCR Program:
“Colony PCR”
1 Cycle
94˚ 3 minutes
25 Cycles
94˚ 30 seconds
55-60˚ 30 seconds
72˚ Amplicon specific (~ 1 minute per kb)1 Cycle
72˚ 7 minutes
4˚ Forever
