Problems with ligation after GeneCleaning

Steven Goldberg goldberg at bms.com
Mon Aug 19 13:03:16 EST 1996


We have been having a problem apparently due to GeneClean and I am
wondering if others have been seeing it too.  After PCR, we remove salts
and clean up the fragment using phenol:CHCl3, precipitate, then perform a
digest to cleave restriction sites on the end of the fragment.  The
reaction is electrophoresed on a TAE agrose gel and the fragment is cut
out and purified using GeneClean.  We have been getting good yields of the
fragment, but it has been impossible to get it to ligate into the
appropriately digested plasmid vector (which was also cleaned up with
GeneClean).  Since we are digesting with two different enzymes (such as
EcoRI and XhoI), in theory the vast majority of transformants should
contain a vector plus insert, but we only get some form of the vector
alone.

The reason that I say GeneClean is responsible for this phenomenon is that
in frustration we performed the same experiment above except GeneClean was
not used to clean up the PCR or restriction digests, and in this instance
10/10 colonies picked contained the correct insert!  Yes, we did purchase
a new kit and made up fresh wash buffer, but this did not correct the
problem. 

We will be using an alternative gel purification kit from now on, but I
wanted to inform other in case they have been seeing the same thing.  If
you have, I would be interested in hearing from you.

Thanks.

Steve



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