IUBio

ClonTECH Site directed Mutagenesis

simon mbsrt at seqnet.dl.ac.uk
Mon Mar 8 17:14:07 EST 1999


On Wed, 03 Mar 1999 15:07:31 -0500, Meg Kawecki <mak9a at virginia.edu>
wrote:

>I have heard that Stratagene Quickchange has been a user friendly kit in
>many hands.  Does anyone have any experience with the Clontech kit?  If
>so, could you let me know at mak9a at virginia.edu.  Thank you.  I have
>been having a lot of trouble with it and I suspect that it could lie in
>my primers simply being a little more picky than the controls supplied
>with the kit.
>Thanks
>Meg Kawecki

I don't think the ClonTech (Transformer?)kit is particularly picky
about the design of primers as long as you follow the rules laid out
in the manual. When  I've had to design my own selection primer I've
had most success  if the enzyme cuts well in the reaction buffer
rather than relying on precipitation/salt adjustment after the
synthesis reaction.

To get the kit to work well you really have to have a very high
transformation effiiciency at the first transformation stage (1x108per
microgram or above) and good selection.   







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