plasmid repair

Johns053 at Johns053 at
Fri Jun 2 10:13:49 EST 2000

I do recall that when I did the such mutagenesis some 15 years ago that the
yield of mutants was rather low and that is why you had to screen via plaque
hybridization.  I found that when I cut the synthesized heteroduplex with a
restriction enzyme and ligated the resulting DNA to a plasmid (which would have
had both strands methylated) that I could skip the hybridization step and find
the mutants by sequencing miniprep DNA.  The efficiency of mutagenesis went from
1-2% to 10-50%.

I pretty sure that single-stranded phage DNA is methylated.

Stewart Johnson

"Dr. Duncan Clark" <Duncan at> on 06/02/2000 10:29:38 AM

Please respond to "Dr. Duncan Clark" <Duncan at>

To:   methods at
Subject:  Re: plasmid repair

In article <852568F2.0049EDDE.00 at>,
Johns053 at writes
>You should check out the papers from Radman's lab in the 1980s which cover
>Repair occurs before replication.  Which strand gets repaired depends on their
>state of GATC methylation.  The non-methylated strand is reverted, for reasons
>of repairing replication errors.  If both strands are methylated or
>unmethylated, the distribution of reversion is 50:50.

A further question on this.  Based on the above, because standard (well
it used to be years ago!) M13 site directed mutagenesis would have given
a mismatched unmethylated strand and presumably the original ssDNA
template strand was methylated(*) how come you got mutants? Or is it the
case that the mutation frequency was very very low but you still managed
to find the mutant by probing.


* I'm unsure whether ss M13 DNA gets dam methylated or not. ds
replicative form will be methylated but does dam methylase, methylate
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382


More information about the Methods mailing list