plasmid repair

Johns053 at mc.duke.edu Johns053 at mc.duke.edu
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 nospam.demon.co.uk> on 06/02/2000 10:29:38 AM

Please respond to "Dr. Duncan Clark" <Duncan at genesys.demon.co.uk>

To:   methods at hgmp.mrc.ac.uk
cc:
Subject:  Re: plasmid repair


In article <852568F2.0049EDDE.00 at Othello.mc.duke.edu>,
Johns053 at mc.duke.edu writes
>You should check out the papers from Radman's lab in the 1980s which cover
this.
>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.


Duncan

* I'm unsure whether ss M13 DNA gets dam methylated or not. ds
replicative form will be methylated but does dam methylase, methylate
ssDNA?
--
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
http://www.dnamp.com
http://www.genesys.demon.co.uk









---






More information about the Methods mailing list