Cloning PCR fragments amplified from a plasmid template

Ashok Aiyar axa12 at po.CWRU.Edu
Fri Oct 4 00:05:35 EST 1991


A friend of mine has been trying to amplify a portion
of a plasmid and then clone that.

He started with 50 nanograms of uncut plasmid DNA as 
template in the amplification reaction.  Following 
amplification, he blunted the PCR reaction with T4 DNA 
polymerase.

He then took 10 ul of the PCR reaction and tried to
blunt end clone it into SmaI digested pUC-19.  He got
dozens of white colonies - most of which turned out
to contain plasmids much larger than expected.

After much speculation on what they could be, he has
now discovered that all of them are the original plasmid
that he did his amplification from.

So I guess that 5 nanograms of supercoiled plasmid (that
has been subjected to 35 rounds of heating and cooling),
transform more efficiently than several molar excess of
ligated plasmid.  I thought this was interesting.  Of 
course, the solution is that it is best to gel-purify a
PCR fragment.  And if plasmid DNA is to be the template,
make sure that it is cut - and use as little as possible.

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