In article <37010135.E078999D at cellbio.unige.ch>,
toufik.abbas-terkit at cellbio.unige.ch wrote:
:: Does any one have a protocol for random mutagenesis by PCR?
: I tried with 1mM of dCTP/dTTP/dGTP and 0.2mM of dATP, but my
: amplification didn't work. (the standart amplification works well!).
:: Thank you for your help
Firstly, when you try to do random PCR by forced misincorporation of
nucleotides it's more common to have 1 dNTP in excess of others, rather
than one lower than the rest. Still the mutations one gets are far from
being random. One very efficient method of mutagenesis that yeilds
mutations of all kinds w.out hotspots is based on substitution of Mg++ by
Mn++. From my personal experience I've found that adding MnCl2 to standard
Taq buffer rather than substituting MgSO4 altogether gives better results.
Better in the way that the frequency of multiple subtitutions is much lower
when one has both Mn++ and Mg++ around, in presence of Mn++ alone a lot of
mutant clones contained double and triple mutations. We (Svetlov and
Cooper) had a little note to that effect in Yeast a year ago. Email me if
you have trouble finding this obscure yeast-data-clearing- house of a
V. Svetlov, Ph. D.
McArdle Laboratory for Cancer Research
University of WI, Madison
1400 University Ave.
Madison, WI 53706