random mutagenesis

Bob's lab pdxsmi at pdn1.gene.nottingham.ac.uk
Fri Sep 27 11:23:50 EST 1996


Dear Stephen,

I've tried several methods for random mutagenesis, but many fail becuse
they produce too many mutations per gene. You could vary the PCR
conditions, by decreasing or increasing magnesium concentrations, or by
using maganese as a substitute. Or you could try limiting a particular
dNTP, and use dITP (ref Spee et al., (1993) Efficient random
mutagenesis method with adjustable mutation frequency by use of PCR and
dITP. Nuc.Acids Res. 21 No.3 pp777-778.)
The best way I found was to use Taq, and rely on its high 'error-rate'
(random misincorporation of a particular base), and then to select for
these mutations. This is done by ligating PCR products, transforming,
and selecting those that fail to complement a strain that carries the
defective gene of interest. I hope this makes sense, Good luck!  Stuart



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