Q: Gel extraction procedure
rsteinbe at etowah.ouhsc.edu
Tue Nov 11 19:44:41 EST 1997
Guy Tremblay wrote:
> I am using a kit for gel extraction of DNA bands. This kit, although
> packaged in a beautiful box, has low yields and I am now inclined to
> throw this box away - even though it has so nice colors. I wish they
> put their effort on something else than the packaging.
> So does anyone have a clean method to do gel extracts that have good
> yields, some kind of an old fashion way that would be clean enough for
> blunt-ended ligations? Or even a kit with high yields and an ugly
> Thanks in advance and have good experiments (without kits).
> Guy Tremblay
> Universite de Montreal
> tremblgu at magellan.umontreal.ca
We gel-purify restriction-digested plasmid DNA and PCR fragments for
ligation all the time using low melting temperature agarose (or NuSieve
GTG for small fragments)-- we don't try to isolate the DNA from the
agarose-- we simply cut out the bands after staining with 0.5 ug/ml
ethidium bromide (taking care to protect from light and using minimal UV
exposure to photograph and/or cut-- we are now using ~3 mM guanosine in
our buffers to protect the DNA as per an earlier thread in this group),
melt the agarose in a 65oC water bath (estimate concentration from input
DNA and volume), and mix fragments to give a total volume of 10 ul for
ligation-- we then add buffer and ligase in another 10 ul and incubate.
This comes out of an old BioTechniques paper by K. Struhl (Vol 3, pp.
452-453 ) that claims that the agarose actually promotes
ligation-- It is important to (melt and) dilute the ligation mixture
about 5-fold before transforming to prevent inhibition.
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