Adding Restriction sites to Primers

Anita Gould anita at accord.cco.caltech.edu
Sat Mar 26 18:03:51 EST 1994


   In article <940309084116.2401704d at vax1.utulsa.edu>, BMA14432 at vax1.utulsa.edu writes:
   > Hello netters,
   > I've picked a couple of primers to PCR my target and now wouuld like to
   > add restriction sites to the 5' ends of them. What are the
   > pitfalls, if any, to be avoided in doing this. Any helpful hints or
   > anecdotes will be greatly appreciated! Thanks in advance
   > 
   > Mark Brudnak
   > University of Tulsa
   > BMA14432 at VAX1.UTULSA.EDU
    
One of my labmates just went thru a lot of grief with this, for
reasons that were obvious in retrospect, but since you're asking for
pitfalls: make sure your oligos are clean, full-length product.  For
20-mers it's not so much of a problem, but when you get out to
50-mers, the chances of any given oligo molecule being full-length can
be unacceptably low (due to the error rate during the synthesis
chemistry, where an error yields a truncated product).  Your synthesis
facility/company might or might not purify the desired product as a
matter of course, so be sure to check w them, & request it explicitly
if necessary.  There are at least 3 choices to purify the desired
product away from the crud:

1) Preparative PAGE
2) HPLC
3) make the last base in the chain a modified nucleotide that can be
pulled out with an affinity column.

My labmate has just ordered new oligos with option #3, which certainly
looks like the easiest.

Best of luck!
--

                                   -Anita Gould
                                    anita at cco.caltech.edu



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