Q: Gel extraction procedure

Chris Boyd chrisb at hgu.mrc.ac.uk
Tue Nov 11 08:34:18 EST 1997


Guy Tremblay (tremblgu at medcn.umontreal.ca) wrote:

: 	So does anyone have a clean method to do gel extracts that have good
: yields, some kind of an old fashion way that would be clean enough for
: blunt-ended ligations? Or even a kit with high yields and an ugly
: packaging?

As an antidote to kits, try the old fashioned freeze-squeeze method
(1). It never fails.  However, in my experience you rarely get more
than about 50% yield, and routinely get 10-30% -- like any other method
(and I have tried many). You just have to live with this. A useful
modification is to plug the lab-made minicolumns with aquarium wool
instead of silanized glass wool (2). Ethanol precipitate as usual, but
for greater efficiency use linear polyacrylamide as carrier (3).
Finally dissolve DNA pellet in TE, and drop dialyse to remove trace
salts and EtOH (4).

There can be problems caused in the gel itself when examined by UV
light.  Avoid DNA damage by minimizing UV exposure and including
guanosine in the running buffer (5).

Of course some of these methods are over five years old and therefore
cannot possibly work as well as the $499 KewlDNAnow!(TM) kit from
SapYourGrant Inc.
 
1. Tautz, D. and Renz, M. (1983) `An optimized freeze-squeeze method for
   the recovery of DNA fragments from agarose gels.' Anal. Biochem., 132,
   14-19.
2. Levine, R. A. (1994) `Aquarium filter floss:  an alternative to
   silanized glass wool as a porous support matrix.'   BioTechniques, 17,
   67.
3. Gaillard, C. and Strauss, F. (1990) `Ethanol precipitation of DNA with
   linear polyacrylamide as carrier.' Nucleic Acids Res., 18, 378.
4. Marusyk, R. and Sergeant, A. (1980) `A simple method for dialysis of
   small volume samples.' Anal. Biochem., 105, 403-404.
5. Gr"undemann, D. and Sch"omig, E. (1996) `Protection of DNA during
   preparative agarose gel electrophoresis against damage induced by
   ultraviolet light.' BioTechniques, 21, 898-903.

Best wishes,
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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