Purifying DNA from gel slices - Summary
vvsvetlov at utmem1.utmem.edu
Thu Dec 12 20:41:25 EST 1996
In article <Pine.OSF.3.95.961212141348.22748A-100000 at sable.ox.ac.uk>,
Alison Reavley <uzdb0017 at sable.ox.ac.uk> wrote:
> DEAE membranes - After running a gel a cut is made just under teh band of
> interest and a piece of DEAE membrane is inserted into the cut, the gel is
> then run an additional 10-15 minutes. The DNA binds to the membrane. The
> membrane is then removed and washed in TE with 100mM NaCl twice to remove
> residual agarose. The DNA is eluted by incubation in TE with 1M NaCl
> overnight ar RT. After elution the sample can be ethanol precipitated.
> Expected recovery 30-50%.
Two points - to anyone working with DEAE paper (DE81) I strongly recommend
to pass eluate through the gel-sizing column (G25, Chromaspin etc) or
extracting with PCI to get rid of the paper fibers (in case of PCI they
stick to the interface). If elution is done at 65oC recovery efficiency
platoes after an hour.
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