Subcloning from polyacrylamide gels
J.Parkhill at bham.ac.uk
Wed May 18 06:47:11 EST 1994
In article <1994May17.194822.1 at samba.cnb.uam.es>,
jenfedaque at samba.cnb.uam.es wrote:
> Does anyone know a simple and reliable method of subcloning DNA fragments
> form acrylamide mini-gels?
> I used to do it in agarose gels, but for small fragments, PAGE's resolution is
> better, so...I wonder if there is a convenient way to recover the DNA.
> (Please, without time-spending electroelutions...etc.)
The easiest method I know is to break up the acrylamide gel slice in an
eppendorf, add 250 ul of EREB, and shake at 37 degC overnight. In the
spin out the acrylamide, wash with more EREB, Phenol,Chlouroform extract
the supernatent and ppt with NaOAc and propan-2-ol.
I find that DNA purified from acrylamide is much easier to subclone than
purified from agarose. This protocol will work with DNA up to 1.5kb.
EREB - (Earl Ruley Elution Buffer)
0.5 M Ammonium acetate
10 mM Magnesium acetate
0.1 mM EDTA
Julian Parkhill - J.Parkhill at bham.ac.uk
CRC Laboratories, Dept. of Cancer Studies, Medical School,
University of Birmingham, Edgbaston, Birmingham, B15 2TJ, U.K.
Disclaimer: If I thought anybody gave a damn about my opinions....
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