fragment isolation problem

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Sun Nov 22 01:17:23 EST 1998


In article <737obm$pfn$1 at mserv2.dl.ac.uk>, "Francisco Villamon"
<fvillamon at hotmail.com> wrote:

> Hi all!
> I want to isolate one fragment to cloning.....the problem is the 
> separation of  one fragment about 2.4 kb from another one about 2.9 kb 
> in an agarose gel. The difference beetwen both after running is so short 
> making difficult the isolation by gel cutting.
> What can I do for separate more the fragments?
> Thanks in advanced,
> Francisco Villamon Cifuentes

Normally one way of achieving this would be to digest with an
enyzyme that cuts the fragment that you don't want (or if
both fragments are required to choose one enzyme per fragment
and use these in separate incubations/separations) and then
separate the intact good piece from the smaller fragments.
Is there any reason that you can't do this?

If you just need enough for subcloning you can run the gel
as best you can (a long, fairly thin 1% agarose gel will work)
making sure not to overload it and then excise the leading or
trailing edge of the band as appropriate (you should actually
get decent separation) and then be prepared to screen any
ligation products to elminate the ones you don't want.

     Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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