best purification of 186bp DNA fragment from agarose

Paquette Yves paquetty at ERE.UMontreal.CA
Fri Nov 25 11:21:51 EST 1994


pnh at ncifcrf.gov writes:

>Robin Colgrove (robin at alumni.caltech.edu) wrote:

>| I am cloning a small (186bp) PCR product. Usually I clone larger fragments
>| and purify the gel fragments with "geneclean." Their package insert
>| suggests not using it for less than 200bp. In the old days, I used all
>| the standard tricks: Low melt agarose, electroelution, mashing up the 
>| gel slice, etc. I was just wondering what people tihink is the best way
>| to go about it these days. Thanks.

>I say go ahead with GeneClean. I've heard from many people that the recovery
>is highly dependent on the specific sequence. I was able to subclone a 35
>bp fragment eluted from glassmilk on the first try, but I started with alot
>of DNA. For other ideas, you could look at the review article I recently wrote.

>@article{Hengen1994Septibs, 
>author = "P. N. Hengen",
>title = "Methods and Reagents - Recovering {DNA} from agarose gels",
>journal = "Trends in Biochemical Sciences",
>volume = "19",
>number = "9",
>pages = "388-389",
>month = "September",
>year = "1994"}

I have used NA45 paper from Scleicher and Schuell to isolate a 50 bp
fragment from an agarose gel, with good yield. You have to be careful
when you precipitate it with ethanol; its better to use a carrier
(like glycogen) and to spin for a longer time than usual (~30 minutes)
in order to recover most of your fragment.
However, I have had a good yield with my 50bp fragment without carrier.
For fragments smaller than 200 bp, you could also use Mermaid (sold
by Bio 101 also). It works great with oligonucleotides for example.

Yves Paquette
Centre de recherche en reproduction humaine
Universite de Montreal
Quebec




More information about the Methods mailing list