Jenny Gipson 10-23-2002
Taq PCR PROTOCOL
1. For a 25ul rxn:
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Use 1ul of 60ng/ul or 100ng/ul DNA
Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each
primer at 100ng/ul concentration
2.5ul 10x PCR Buffer w/ Mg (1.5mM)
0.5ul 25mM MgCl2
0.5ul dNTP
0.125ul Taq
18.375ul sterile water to equal a 25ul rxn
(*if not making master mix, dilute Taq so that you can add 1ul of Taq and 17.5ul
sterile water to equal a 25ul rxn)
2. For a 50ul rxn:
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Use 2ul of 60ng/ul or 100ng/ul DNA
Use 2ul of each primer at 3.2pmole/ul concentration or 2.5ul of each
primer at 100ng/ul concentration
5ul 10x PCR Buffer w/ Mg
1ul 25mM MgCl2
1ul dNTP
0.25ul Taq
36.75ul sterile water to equal a 50ul rxn
(*if not making a master mix, dilute Taq so that you can add 1ul of Taq and 36ul
sterile water to equal a 50ul rxn)
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Keep the reagents on ice.
Add the Taq last, and keep it in the freezer until you are ready to add it.
Vortex briefly and quick spin.
Cycle:
 95C for 1-5minutes (usually 4min)
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95C for 1min
55C for 1min
72C for 1.5 to 2min (usually 2min)
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72C for 10min
4C hold
Cycle 30 times