Subcloning Blues

Dr. Arthur L. Kruckeberg bmbckh at biovax.leeds.ac.uk
Wed Feb 7 12:52:04 EST 1996


Dear Netters,

I have been experiencing very frustrating subcloning blues.  I have
PCR'd
1-kb DNA fragment with PstI restriction site and extra 2-bp at each
end. 
Then, the fragment was agarose gel-purified, digested fully (I tried
one-
and two-overnights), and gel-purified again.  I tried ligation of this
fragment to a vector of 7.3 kb which was completely cut with PstI and
dephosphorylated.  I should have positive clones, but got nothing -
there
was even no antisense ligation! 

I'm sure that PstI restriction enzyme, ligase, and transformation are
working good.  I suspects the DNA fragment might not be cut even though
it
has been treated thoroughly.  When I tried to make concatemer formation
with self-ligation of the PstI-digest of the fragment, I got only the
same
1-kb band like the unligated - no band around 2-kb at all.

According to NEB, PstI-site-containing fragment with extra 2-bp's
should be
cut over 90% after an overnight.  However, is there any possibility
that my
fragment was not cut and I hasseled around with this uncut fragment?  I
would really appreciate any comments about this.  Please e-mail Namjin
Chung at chung006 at mc.duke.edu  


Namjin Chung

Why not blunt-end ligate instead?
------------------------------------------------------
Chris Hoyle

   Department of Biochemistry and Molecular Biology
University of Leeds             phone +44 0113 2333172
Leeds LS2 9JT                   FAX   +44 0113 2333167
Great Britain           e-mail bmbckh at biovax.leeds.ac.uk
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