DNA purification from agarose gels

[andy chien]

Our lab does not use any of these kits for the purification of 
DNA fragments from gels.  Instead, we use a technique that was 
recently described in Biotechniques.  What we do is we run our 
DNA sample, at 50-100 mV for a 10 cm gel, and as soon as the 
band is well resolved, we use a sterile razor blade or scalpel 
to carve out a  well immediately in front of the band of 
interest.  The closer the well is to the band, the easier it 
will be to isolate.  The well is usually about 1 cm. long.  
Then we lower the buffer in the running chamber so that it is 
just below the level of the gel, and not spilling over the top. 
 Then we fill the newly-cut well with 200-400 ul of running 
buffer, and crank the gel at 150 mV for 1-2 min.  Then we pipet 
out the band, which has run into the well, which we can also 
track by UV.  You can do this repeatedly, adjusting the time of 
elution according to the size of the fragment, and after 1-6 
runs of eluting and re-filling the well and re-eluting, you can 
get good recovery of DNA.  This DNA can be pptd. with acetate 
and ethanol, and subsequently used directly for ligations.  We 
have great success with this technique, as do many other labs 
on our campus.  You can use GeneClean on the eluted product if 
you want, but we don't find that to be an essential step.  Our 
yields are between50-100% for large (>4kb) products, and near 
100% for products < 4kb.  This would save you a lot of time and 
money.  Some people also add PEG to the well, but we don't.  
For further reference, check Biotechniques, or you could e-mail 
me.