Questions About the Termini of PCR Products

Ashok Aiyar axa12 at po.CWRU.Edu
Fri Sep 27 13:38:32 EST 1991


Even when I have had restriction sites introduced by the primers,
I have personally not had much success cloning PCR products.

I have routinely started doing the following to a 100 ul PCR
reaction.  Cloning results have been a lot more reproducible and
are better.

After PCR reaction, I add 5 units (1 ul) of T4 DNA polymerase,
and 1 ul of 300mM MgCl2.  Incubate at 37 degrees for 30 minutes.

I know gel purify one-third of this reaction by electrophoresing
onto DEAE-nylon membrane (sold by S&S).  After eluting the DNA with
high salt, I blunt end clone it into the SmaI site of pUC18 or
Bluescript.  After getting the right clone, I subclone from
pUC or from Bluescript into the vector of choice.  I do normally
phosphatase treat the vector.

Even when restriction sites have been 5 bases internal to the 5'
end of the primer, I have had no luck cloning the PCR products
directly.  I have never tried a restriction digestion after
treating with T4 DNA polymerase.

Regards,

Ashok Aiyar
Department of Biochemistry
CWRU Medical School
-- 
  Ashok A. Aiyar   Department of Biochemistry   CWRU Med. School
   		       axa12 at po.cwru.edu
		   aiyar at cwbio.bioc.cwru.edu
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