Error Prone PCR

Materials and Reagents

Mix

  • 1 μL of dilute DNA to be mutated (10’s of fmol)

  • 1 μL of 25 μM forward primer

  • 1 μL of 25 μM reverse primer

  • 4 μL of 5 mM dGTP

  • 4 μL of 5 mM dATP

  • 20 μL of 5 mM dCTP

  • 20 μL of 5 mM dTTP

  • 1 μL of Taq Polymerase (5 units)

  • 5 μL of 10 mM MnCl2

  • 3 μL of H2O

  • 40 μL of 2.5x buffer

2.5x Buffer

  • 125 mM KCl

  • 25 mM Tris-HCl pH-8.3

  • 0.03 % by mass BSA

  • 17.5 mM MgCl2

Procedure

  1. Add MnCl2 after all the other PCR components except for Taq to prevent precipitation.

  2. Prepare a total reaction buffer of 100 μL.

  3. Run 30 cycles of denaturing, annealing, and elongating.

  4. Finish with a 10 minute elongation period at 72 degrees Celsius.

Notes and Cautions

  • Concentration important for final error rate.