DNA purification from Agarose (Sigma)
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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