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.