Unequal dNTP mutagenesis
Principle: unequal dNTP concentrations and high MgCl2 concentration forces disincorporation of dNTPs
Recipe:
25uL H2O
5uL 10uM primers
5uL 10X buffer
1uL template (20ng)
1uL 10mM dATP
1uL 10mM dGTP
5uL 10mM dCTP
0.5uL 100mM dTTP
5.5uL 50mM MgCl2
1uL Taq
Mike achieved 3-4 amino acid substitutions per 3kb with these conditions. He also saw variable amplification depending on which Taq he used, but I've had good amplification with NEB Taq.
MD
10-18-12