Best gel extraction kit?

[Kajika]

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I've been using QIAquick spin columns for gel filtration of PCR fragments on the order of 150 base pairs, but my yield has been terrible (~10%), and others in my lab have noticed when doing atomic force microscopy that there is residual agarose and resin along with the eluted DNA. What gel extraction kits do you use, and what kind of results have you had?

 

[DrChandy]

I've been using QIAquick spin columns for gel filtration of PCR fragments on the order of 150 base pairs, but my yield has been terrible (~10%), and others in my lab have noticed when doing atomic force microscopy that there is residual agarose and resin along with the eluted DNA. What gel extraction kits do you use, and what kind of results have you had?

What do you use the PCR product for? Depending on the use of the product, a low yield may not be an issue. I use QIA quick spin columns for subclonings, and the concentration of DNA from the gel isolate is fine for ligation reactions. Volumetric yield from the spins is approximately 30-40 microliters per spin. Concentration of DNA from the isolate varies from my experience. It may help to finely chop the isolated fragment before spinning- this maximizes yield. Also, freezing the fragment for 5 minutes in a -20C freezer before spinning it at room temperature helps maximize the yield.

 

[DrChandy]

You can also use a lower percentage gel to increase DNA yield. You could use a 1% gel (using 1.0 grams of agarose in 100ml Tris Boric Acid EDTA [TBE]), which results in more DNA per gel isolation preparation, as less DNA can be retained in the agarose after your spin. It's good to use a 1.5% gel for gel isolations, however, so as to be safe from losing the fragment in case you over-run the gel.