Blunt-end troubles

John Watson watson_j at bms.com
Thu Mar 6 08:04:58 EST 1997


Andrew Doherty 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?
> > Cheers,
> > Tim ;-)
> The only thing I can think of is that the insert is lethal when it's the
> right way round - other than that, I'm buggered if I know!!!!
> Hope it helps

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.

----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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