Subcloning Blues

Namjin Chung chung006 at
Fri Feb 2 11:46:42 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  

Namjin Chung
Program in Molecular Cancer Biology
Department of Cell Biology and Medicine
Duke University Medical Center

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