Jenny Gipson 10-23-2002 PCR PROTOCOL

1. 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

(*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: 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) 3. 4. 5. 6. 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: 95C for 1-5minutes (usually 4min) 95C for 1min

Jenny Gipson 10-23-2002 55C for 1min 72C for 1.5 to 2min (usually 2min) 72C for 10min 4C hold Cycle 30 times