Mutagenic PCR

[Jerome.Crittin at unil.ch]

I am currently trying to perform random mutagenesis of a
gene but the PCR does not work! I recently read a protocole
about that (found on the internet) which was as follows:

10 microliters of 10x error-prone buffer*
10 microliters of DMSO
3 microliters of 10 mM MnCl2
50 pmol of each primer
10 ng of template DNA
dNTPs (dATP & dGTP to 0.2 mM, dCTP & dTTP to 1 mM)
5U of Taq polymerase
H2O to 100 microliters

*100 mM Tris-HCl, pH=8.3; 500 mM KCl, 70 mM MgCl2, 0.1%
gelatin

I tried to vary some parameters like [MgCl2], [DMSO],
[primers], but my PCR does not work at all! The program i
use is correct, since i tested it with a standard PCR
protocole, using the DNA, primers,...i use for mutagenic
PCR. Do some of you have any tips or suggestions to make my
random mutagenesis work?

Thank you