Blunt-end troubles

Barry Mook BarryMook at aol.com
Wed Mar 5 12:27:40 EST 1997


In article <5fhd02$8to_001 at leeds.ac.uk>, BMBTJY at leeds.ac.uk (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 ;-)

If you are making a single cut in the MCS then you will always get 2
orientations...but you can make a double cut....:

-cut the MCS with one blunt cutter...CIP the vector, then cut with the
second enzyme....this way only one side will be phosphorilated....do the
same to your insert and hoppa...it can only go in one way

- if worse come to worse..use linkers to make the blunt ends sticky

good luck...it can be a b---- sometimes.

Barry



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