PCR PROTOCOL

For a 25ul rxn:

·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 ~undefinedif not making master mix, dilute Taq so that you can add 1ul of Taq and 17.5ul sterile water to equal a 25ul rxn) For a 50ul rxn: 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 ~undefinedif not making a master mix, dilute Taq so that you can add 1ul of Taq and 36ul sterile water to equal a 50ul rxn) 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)

·95°C for 1min

·55°C for 1min Cycle 30 times

· 72°C for 1.5 to 2min (usually 2min)

·72°C for 10min ·4°C hold