Problems with ligation after GeneCleaning

Nori Shiraishi aa47093 at hongo.ecc.u-tokyo.ac.jp
Sun Aug 25 04:22:21 EST 1996


In article <goldberg-1908961303160001 at 140.176.74.203>, goldberg at bms.com
(Steven Goldberg) wrote:

> 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.
> 
Isn't that the contamination of the thermostablr polymerase(like Taq)
you used?? Some one in my lab says that the contaminating polymerase
works in the 
TAE agarose gel, and the restricted DNA fragment was filled in.
Try change the buffer of the electrophoresis everytime you use.

thanks



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