Blunting sheared DNA

Chris Boyd cboyd at holyrood.ed.ac.uk
Mon Jan 15 04:43:17 EST 2001


: At 03:14 PM 1/12/01 -0800, Chris Upton wrote:
:>Hi, we're going to be generating randomly sheared DNA for sequencing using
:>a nebulizer, and cloning into an Invitrogen Topo-Blunt vector.
:>
:>Anyone have any suggestions for blunting the ends?
:>We have some of the Stratagene  PCR polishing kit around that uses Pfu.
:>Anyone tried this to fix sheared DNA?

"Dr. Hiranya S. Roychowdhury" <hroychow at nmsu.edu> wrote:
: Never done with sheared DNA, but I would think that T4 DNA polymerse would
: work with its 3-5' exo and 5-3' poly activity.

... remembering to add an excess of dNTPs with the polymerase.

Best wishes,
-- 
Chris Boyd                      | from, but \    Medical Genetics Section
Chris.Boyd at ed.ac.uk             | not for,  /    MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd                       EH4 2XU, SCOTLAND
NEW! transgenics / CF gene therapy job:  http://jobs.ac.uk/jobfiles/HH286.html






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