Taq 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)

  1. 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)

  1. Keep the reagents on ice.
  2. Add the Taq last, and keep it in the freezer until you are ready to add it.
  3. Vortex briefly and quick spin.
  4. 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