DNA from Gel - GeneClean

Bob Rutledge brutledge at pnfi.forestry.ca
Mon Mar 28 20:44:19 EST 1994


In article <2lq4ea$kh0 at quartz.ucs.ualberta.ca> asimmond at gpu.srv.ualberta.ca (Andrew Simmonds) writes:
>From: asimmond at gpu.srv.ualberta.ca (Andrew Simmonds)
>Subject: Re: DNA from Gel - GeneClean
>Date: 11 Mar 1994 15:58:02 GMT

>Tony Hodge (tph at mrc-lmb.cam.ac.uk) wrote:
>: Subject: Isolating High MW DNA from Gel- Best Way??
>: From: Peter T. Boag, BOAGP at QUCDN.QUEENSU.CA
>: Date: Thu, 10 Mar 1994 17:31:03 GMT
>: In article <CMGLzs.Isz at knot.ccs.queensu.ca> Peter T. Boag,
>: BOAGP at QUCDN.QUEENSU.CA writes:
>: >We are doing some genomic library building and want to
>: >preferentially clone fragments in the 5 to 15 kb range. We have
>: >experimented with various ways to isolate this range of DNA from an
>: >Agarose gel, and always seem to end up with a low yield. We have
>: >tried inserting  a piece of dialysis membrane and running the DNA
>: >onto it. We have cut a well in the gel ahead of the desired area,
>: >then pipet off the DNA as it runs into the well.  We are thinking
>: >now of excising the relevant piece of gel and spinning it above a
>: >siliconized glass wool plugged       eppindorf with a hole in the bottom
>: >inserted into a second eppindorf, and collecting the runoff (this is
>: >a variant of the "freeze and squeeze" approach).
>: >
>: >Any suggestions for this type of isolation, either quit and dirty or
>: >fancy commercial approaches such as these electroelution chambres??


>: I always found GeneClean from BIO 101 to be the ultimate in DNA
>: recovery from agarose, I have used it or a variant for many years. 
>: Recently I have had problems with the latest version, GeneClean II,
>: but this seems to be due to the High Salt buffer going off after 6
>: to 12 months, buying a new kit when this occurs clears up the
>: problem.  The principle is that saturated NaI (or perchlorate in
>: some systems) both melts the agarose at elevated temperature and
>: (due to high salt) causes the DNA to adhere to powdered glass (or
>: similar in other kits).  the DNA/Glass is washed extensively and
>: then the DNA eluted off with low salt buffer or water.

>: The kit can be used for recovery of DNA from agarose, de-salting,
>: removing eg restriction enzymes prior to ligation, cleaning up PCR
>: products and curing the common cold, try it and see.



>: Tony




>One problem with Geneclean is that the glass fines tend to break up large 
>fragments of DNA due to the DNA binding to multiple glass beads and 
>basically being pulled apart during re-suspention of the glass/DNA 
>mixture...(I seem to remember a disclaimer to that fact on the Geneclean 
>documentation).  One improvement I have seen on this technique is to use 
>a cartridge/insert, (orginally developed by Millipore?) that is basically 
>a glass filter arranged in such a way that you can run the DNA mixture 
>through it and have it adhere without the problems associated with a 
>glass powder.  It is marketed as a kit in Canada under such names as 
>Glassmax(Gibco/BRL) etc.
>(I don't work for either firm, just use their stuff....)  
>______________________________________________________________________________
>Andrew Simmonds - Department of Genetics - University of Alberta CANADA
>ASIMMOND at GPU.SRV.UALBERTA.CA
>ASIMMOND at VM.UCS.UALBERTA.CA
>G216 Biological Sciences Centre
>Edmonton, Alberta, CANADA T6G 2E9
>******************************************************************************
>            "Does steel wool come from magnetic sheep?" 
>******************************************************************************

We have successfully constructed several genomic libraries using GeneClean for 
preparing18-24 Kb fragments following agarose electorphoresis of partially 
digested gDNA.   Little if any fragmentation was apparent as assessed by 
reelectrophoresis, and subsequent ligations produced high titers.

Bob Rutledge
Petawawa National Forestry Institute
Canadian Forest Service   



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