SOS!--PCR restriction cut

Ashok Aiyar aiyar at ebv.oncology.wisc.edu
Mon Mar 18 08:53:46 EST 1996


On 17 Mar 1996 19:17:14 GMT, Wenjin Yu <wyu at uoguelph.ca> 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.

If your PCR product looks clean and sequences appropriately, I suggest
that you try the following:

a) repeat the PCR with phosphorylated oligos (kinased at the 5' end)
b) gel purify the PCR product (away from primer dimers)
c) self-ligate the PCR products (1 hour at 37oC is sufficient)
d) phenol extract and precipitate the ligation reaction
e) Cut the ligated product with your enzymes and gel purify the
   band that corresponds to the linear

I have used this type of procedure several times and it has worked
quite well.  The one caveat is that the PCR product should not have
any non-templated nucleotides at it's 3' end (such as dA).  This can
be avoided if you use Vent/Pfu/Pwo instead of Taq.

Ashok
--
Ashok Aiyar, Ph.D.
Department of Oncology                aiyar at ebv.oncology.wisc.edu
Univ. of Wisconsin-Madison                    tel: (608) 262-6697




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