Blunt-end troubles

Darren Natale dnatale at box-d.nih.gov
Thu Mar 6 13:55:24 EST 1997


John Watson wrote:
> 
> > T.J. Young wrote:
> > >
> > > Hi,
> > > I've got a bit of a problem, I'm trying to subclone a DNA sequence from one
> > > vector into another.  Unfortunatly, the MCS are not compatible so I'm using
> > > blunt-ended ligation.  Now this is a bugger at the best of times but every
> > > time I get some positive colonies, on inspection, I find that the sequence
> > > has gone in the wrong way round!!  Now am I just the Worlds unluckiest
> > > molecular biology student or could there be some kind of underlying
> > > phenomenon that's driving my ligation in this direction?
> 
> I can only add my personal experience that blunt end ligation often seems to favor
> one orientation and not both as might be expected.  I've also found this to be the
> case with T-vector ligations as well.  We recently screened 96 T-vector
> transformants by PCR -- all had the insert the "wrong way" round.  And yes, it did
> make the sussequent subcloning a pain.  I wouold love to know if there is a sound
> biological/physical reason for this apparent phenonmenon.

I have had the same experience with TA cloning.  I can only add this: in
my case, 
the PCR product (insert) contained part of the MCS of pBluescript,
including one of
the M13 primer sequences (in some cases forward, in other cases the
reverse).
Interestingly, in about 90-95% of the clones (white colonies using
blue/white
screening), the insert was oriented such that the M13 sequences faced
each other 
(so that using, say, only the reverse primer, a PCR product would be
obtained).  
That is, the M13 sequences from the insert would form an inverted repeat
with the
M13 sequences from the vector (opposite to what one would expect). 

D. Natale
dnatale at box-d.nih.gov



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