Stratagene QuikChange Problems

Richard J. Dudley rdudley at nrc.uab.edu
Wed Mar 4 14:42:42 EST 1998


Rick Davis wrote:
> 
> Hi All,
>         I'm currently trying to remove a 28 bp intron from my cDNA
> clone utilizing Stratagene's QuikChange site-directed mutagenesis
> kit. I designed primers on opposite strands that flank the intron
> which should have resulted in elimination of the intron after PCR
> amplification. Instead I find that all ten of the isolates that I
> sequenced still have the intron present! I designed the primers
> according to Stratagene's recommendations and used 18 cycles
> of amplification. Has anyone out there used this kit to remove
> introns? Are there any other kits/procedures available that would
> do a better job? TIA

Stratagene's probably already contacted you, but here's my $0.02 worth
anyway.

What it sounds like is that you may not be fully digesting your parental
DNA with DpnI.  Also, was your DNA isolated from a methylating host
strain?

You may want to try purifying your PCR products and doing a blunt-end
ligation, then transforming that ligation.

Good luck!

rich

--- --- --- -- -- -- --- --- ---
* Yes, Kathy really did let me name the cat Histidine. *

Richard J. Dudley (rdudley at nrc.uab.edu)                            
Department of Neurobiology                    
University of Alabama School of Medicine       
http://www.nrc.uab.edu/



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