DNA from Polyacrylamide Gels

Bruce Roe broe at aardvark.ucs.uoknor.edu
Thu Dec 10 08:38:00 EST 1992


In article <ashok.133.723947114 at meds38956.cwru.edu>, ashok at meds38956.cwru.edu (Ashok Aiyar) writes...
>In article <1992Dec9.061342.26494 at kudpc.kyoto-u.ac.jp> 
>	a52041 at sakura.kudpc.kyoto-u.ac.jp (Michael CHENG) writes:
> 
> 
>>  Hi! I was wondering if anyone out there knows of any company that sells a kit
>>for extracting DNA from polyacrylamide gels. According to Molecular Cloning
>>(2nd Edition) by Sambrook et al on page 6.46, there exists a "Crush and Soak"

	<stuff deleted>

For fragments <100 we just cut out the band with a steril blade, place the
INTACT gel piece in a siliconized 0.5 ml microfuge tube.  Add  enough  OGEB
(see below) or water to cover the gel slice (approximately  200 ul)  and 
place at 37deg C for from 3 hours to overnight.  Then pull out the gel piece
with tweezers, do a brief spin to pellet any small gel pieces and run the
supernatant through a small G-25 column (spin columns work fine).  The yield
we get for 20 mers is >50% which is quite sufficient for us, but the yield
via this diffusion method without crush and soak drops to about half that
for larger (200 bp) fragments.  One of the major problems with the crush
and soak method is that the DNA sticks to non-siliconized glass rods that
some use for crushing the gel fragment and that dramatically reduces the
yield.  Improved yields can be obtained via crush and soak by siliconizing
the tubes and other implements.
	OGEB = OligoGelElutionBuffer = 500mM NH4OAc, 10mM MgOAc, 1mM EDTA
Cheers.......bruce
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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