Subcloning Blues

Lena Polovnikova elena at chem.purdue.edu
Tue Feb 6 08:37:26 EST 1996


In article <t.barnett-0402961343530001 at 131.217.101.118>,
t.barnett at path.utas.edu.au (Tim Barnett) wrote:

> In article <4eu2d6$270m at news.doit.wisc.edu>, <@students.wisc.edu> wrote:
> 
> > Hmmm, I have a similar problem, and I've never been able
> > to get it to work.  I'm also using a PCR product with PstI.
> > 
> > If anyone has any suggestions, please post them.  Thanks.
 
> Try a blunt-end ligation to make concatmers of your original PCR product
(you will have to amplify with an enzyme which doesn't add an extra A
residue as Taq does or blunt end Taq-amplified products) and then digest
this with Pst I.  If your restriction site is at the end of a fragment
then it probably won't cut.
> 
I had similar problems with restriction sites on the ends of PCR fragments
and was never able to subclone those fragments directly using this
restriction sites.  When  I ligated the fragments into concatamers and
tried to cut them it still did not work.  So I ligated the PCR products
after polishing the ends into   Sma I site of pBluescript and sequenced
them. It turned out that most of the PCR products were missing several
bases on the ends, so the restriction sites were simply not there (I found
a full-length product eventually).
                                                                       Lena.

-- 
Lena Polovnikova
Purdue University BMB Program
elena at chem.purdue.edu



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