DNA purification from Agarose (Sigma)

Mike Witty m.witty at ic.ac.uk
Thu Oct 12 17:01:54 EST 1995


In article <45fr29$74p at worak.kaist.ac.kr>, hbkim at biochem.kaist.ac.kr
(Hanbok Kim) wrote:

> For DNA purification, Geneclean II Kit (Bio101) with
> TBE modifier and Sigma Agarose (A9539) was used.
> However subcloning was very hard by this method, suggesting
> some inhibitory effects on ligation by either Agarose 
> or Kit ingredients. Ligation as a control was ok.
> I would appreciate any advices to solve the problem, and
> ingredients of TBE modifier.

What I do is use TAE buffer, not TBE, cut out the gel slice and spin it
through an eppendorf tube which has a TINY hole in it, plugged with glass
beads.  I ethanol precipitate the filtrate and resuspend 1 band of about
100ng or less in 10 microlitres of TE.  5 microlitres of vector is ligated
to 5 of insert.  I use ordinary agarose (from Gibco BRL, a UK supplier)
and I have
not had any trouble with ligations.  This method is not very efficient in terms
of percentage yield of DNA, but is cheap and easy.  Good luck, Mike.



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