Problems with ligation after GeneCleaning

Yoonsuk Park yoonpark at u.washington.edu
Tue Aug 20 11:29:24 EST 1996


We are having a same problem.  We have checked the DNA fragment after
elution, and don't think degradation is the problem.
I have used GeneClean for several years, but haven't haven any problem to
subclone DNA fragment using this kit up to now.  This is a new problem for
me.  Does anyone help me to solve this problem?
Thanks in advance!

Yoonsuk.

       >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
                Yoonsuk Park, Ph.D.
              University of Washington
          Dept. of Oral Biology, Box 357132
              Seattle, WA, 98195-7132

         E-Mail: yoonpark at u.washington.edu
      <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

On Mon, 19 Aug 1996, 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.




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