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Team:Washington/PCR GOTAG
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Revision as of 03:44, 21 September 2013
""
PCR using GoTaq
1. Mix reaction:
For 50 ul rxn
25 ul 2X Green Taq
1 ul 10 uM forward primer
1 ul 10 uM reverse primer
1 ul DNA template (10-100 ng)***
22 ul dH2O
2. Run PCR reaction on thermocycler:
95C, 3 min for colony PCR: 30s for Miniprep PCR
95C, 30 sec
50-65C*, 30 sec
72C, 1 min**
Repeat steps 2-4, 35x
72C, 5 min
4C, hold
*Annealing temp should be adjusted for the primers you are using. Typically, 5C lower than the Tm of your primers works great (IDT prints the Tm on the oligo label).
**Extension time should be adjusted based on product length. Taq can polymerize 1 kb/min
***If PCRing insert use 1ng/ul DNA concentration.
