Purifying PCR product from 2% agarose gel

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Mon Feb 19 15:44:41 EST 2007


On Sun, 18 Feb 2007, hilary cassidy wrote:

> I've been trying to purify my PCR product from a 2% agarose gel all week
> now as it needs to be purified prior to Topo cloning. I've used both the
> QIAEX II Gel Purification kit by Qiagen and the Illustra DNA and Gel
> Band Purification Kit by GE Healthcare. Each time I have strong bands on
> the gel prior to cutting the bands, but everytime I purify and run the
> DNA on a gel to check there is nothing!!!

I used to use QIAEX II and had a lot of trouble with it; silica slurry
pellets are not very stable, and i found i tended to either lose bits of
it, or not be able to wash it properly. If you can, try the QIAquick kit
instead, which has a fixed silica membrane; i (and dozens of other people
in my institute) use this routinely with great success. I've never
extracted from a 2% gel with it, but i can't imagine it being a big
problem.

I'm afraid i don't know the Illustra kit.

How new are the kits? They do go off over time - i've had some very
frustrating times trying to troubleshoot a purification when that's
happened.

If nothing else works, try the ghetto elution: cut a 1000 ul filter tip
just below the filter, put the gel slice in the top, put it in a
microtube, and spin it at full tilt until all the buffer's been squeezed
out of the gel (which stays on to of the filter) down into the bottom of
the tube. The yield is nothing to get excited about, but it's foolproof,
and not sensitive to weird chemical goings-on in the sample.

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk



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