Stratagene QuikChange mutagenesis?
rand at pelican.dbe.csiro.au
Tue Feb 11 01:13:53 EST 1997
In article <5dmsja$m1f at bioalp.biobase.dk>, wind at biobase.dk (Troels Wind) wrote:
> Greg (jquinn at nntp.best.com) wrote:
> : A question for those who might use this brand of mutagenesis kit:
> : I am currently having some problems with a high background using the
> : QuikChange kit. Does anyone have any experience in reducing this?
> : Thanks for any help
> This may be stated in the protocol, but anyway...
> You can try the following:
> Increase the amount of DpnI in your digestion and/or perform it
> for longer time. This should reduce background.
> Perform several reactions with varying amounts of parental plasmid.
> I do 2, 5, 10 and 25 ng and pick colonies from the reaction with
> the lowest amount. (The lower the amount of parental plasmid, the
> better the chance that that the DpnI digest is complete.)
> Good luck!
> Troels Wind
It used to be said that DpnI would only cut if both DNA strands are
methylated - I presume that in the Stratagene method the majority of
parental (methylated) strands would be hybridized to the new, unmethylated
strands so cutting may be inefficient.
Or has someone recently shown that DpnI cuts hemimethylated strands efficiently?
Keith Rand, Sydney Australia
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