SOS!--PCR restriction cut

ProZyme info at prozyme.com
Mon Mar 18 21:10:02 EST 1996


Wenjin Yu wrote:
> 
>         I have a big problem to cut PCR product in which the restriction site
> is generated in the primer.  At first I designed a primer containing a Bam
> HI site.  Based on the inforamtion in New England Biolabs Catalog, 4
> extra bases was added at the 5'-end, but the PCR product couldn't be cut
> no matter how much enzyme was used.  Then I made another primer
> containing an EcoRI site, and 15 extra-bases was added to the 5'-end this
> time.  But it seems still not work!  I was wondering if anyone can help me to
> deal with this big trouble.  Many thanks.
>  (please send your HELP to my mail box: wyu at uoguelph.ca)
> Wenjin Yu
> U of GuelphI have cloned PCR fragments with inserted restrictions sites at the ends 
several times and I've had good success.  I've incorporated BamH1 sites 
(among others) and I've found that four extra bases 5' to the site is 
sufficient.  You don't say how you purify your PCR product before 
cutting it; I think this is an important step.  I use the Geneclean kit 
from Bio101 and I follow the "Double Geneclean" procedure.  That is, I 
Geneclean-purify the PCR product, digest it and Geneclean-purify it 
again before ligating.  Hope this helps.

Bruce Amsden



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