SOS!--PCR restriction cut

C.L.L. ChunLin_Lu at cellbio.duke.edu
Mon Mar 18 13:03:17 EST 1996


Normally, I only add 3 extra bases to its 5'-end for most of my
constructions, have not found any problems no matter which restriction
site  I put. It maybe a crucial step that your interested fragents need to
be digested for two days, sometimes I added   additonal enzymes to ensure
reactions, and gel purified fragents before cloning. I cloned ten
different genes into various vectors, all  worked. Hope it helpful.
Chunlin
In article <4ihofq$kvp at ccshst05.cs.uoguelph.ca>, wyu at uoguelph.ca (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 Guelph



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