about DNA recovery from agarose gel

[Jill Kellogg]

on 7/11/96 Jin (jim at U.ARIZONA.EDU (Jin-seon Im) at INTERNET) wrote:

>i have a quick (?) question
>i know there are several ways to recover DNA from agarose gel like
> with glass beads 
>      Qiagen
>      freeze squeeze methods
>and we can use low melting agarose gel
>
>how do you think which one is better to recover the DNA from agarose gel?
>and you purify the DNA from pcr product with those methods,
>then, how about jsut EtOH precipitation of pcr product?
>
>in terms of the recovery, which one will be the best?
>
>any good comments, please

I know this won't answer all your questions but here's one method.

If I know my PCR fragment is a single band, and therefore see no reason to gel
purify, I purify the reaction as follows.

phenol:chloroform:Isoamyl (25:24:1) extract using a phase lock gel (From  5
Prime to 3 Prime, 800-533-5703)  Phase locks gels are not necessary, of course,
but they do improve recovery rates by allowing complete recovery of the aqueous
phase. I do this step first to get rid of the enzyme and any other protein and
junk that might precipitate later when I add salt to the sample.

Run the aqueous phase through a spin column, usually G-50.  This removes dNTP's
and the primers.

Add a co-precipitant.  My favorite right now is Pellet Paint, a color modified
glycogen, from Novagen.  They do the quality control so no matter what I want to
do later with my fragment I know the Pellet Paint won't interfer.  Again a co-
precipitant is not necessary but I can get higher recovery of my fragment in
less time, i.e. 5 minutes on ice, 5 minutes in the microfuge.

Then I add a salt, usually Na-acetate, and ethanol, ice and spin.  Wash the
pellet 2 times in 70% ethanol. Dry and resuspend the pellet.

If you do need to run your PCR on a gel I've been real happy using low melt
agarose (FMC) and AgarACE from Promega.  For an agrase it's fast; no buffer
exchange.



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