what's the best way to clone PCR products?

Tracy Aquilla aquilla at salus.med.uvm.edu
Mon Sep 26 11:06:07 EST 1994


In Article <35v1pa$9rn at steele.ohsu.edu>, nishir at ohsu.edu wrote:
>In article <Lippay-220994132117 at mac02019.sd.gat.com> Lippay at vaxd.gat.com (Eric
>Lippay) writes:
>>
>>> 
>>
>>                                       I have also used Invitrogens TA cloning
kit many times with good
>>results.  The method is straight foreward and reliable.
>>
>>
>>Eric Lippay
>>General Atomics
>>Bioscience Division
>>
>We were happy with the Stratagene PCR Script kit until we ran out of the
>restriction enzyme (way before our vector ran out, of course).  The srf1 is
>very expensive and sold only by Stratagene.  We turned to using sma1, which
>worked until we had a very bizarre thing happen (any explanations are welcome).
> We made PCR primers that encoded a kpn1 site on one end and Xba1 on the other
>and subcloned the product into PCR script with sma1 (we knew our product had no
>sma1 site).  When we tried to cut the vector with insert with kpn1 and xba1, we
>got a larger piece out (larger by 70-80 bp).  Since the vector has a kpn1 site
>about that distance away from the cloning site of the PCR product, we figured
>that the kpn1 site in our product had gotten destroyed, and the enzyme was
>cutting at the kpn1 site in the vector.  Could the blunt cloning with sma1 have
>nibbled back our kpn1 site in the product so that it was no longer there?  BTW
>we double-checked our primers and kpn1 is really there.
>
>Rae Nishi
>
>

    Did you polish the ends of the PCR product with Klenow (or another pol)
before cloning into the SmaI cut vector? I assume you must have. If so, the
KpnI site was probably destroyed by removal of the 4-base 3' overhanging
end. Klenow has 3'-5' exonuclease activity (the 5'-3' polymerase activity
fills in 5' overhangs only), so when you try to "fill in" ends with 3'
overhangs, they are actually deleted, not filled in. This works OK for
cloning, unless you wanted to use that RE site for something. My best guess
is, that was the problem you had.
Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aquilla at salus.med.uvm.edu



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