Stratagene QuikChange mutagenesis?
khuang at CHEMVX.CHEM.TAMU.EDU
Tue Feb 11 13:01:45 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
>Keith Rand, Sydney Australia
I want to ask you guys how many samples do you sequence by using
Quick-change Kit for doing site-directed mutagenesis?
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