Purifying DNA from gel slices - Summary

Alison Reavley uzdb0017 at sable.ox.ac.uk
Thu Dec 12 09:40:47 EST 1996


Recently I posted a question regarding the purification of DNA from
agarose gel slices without the use of organic solvents or increasing the
temperature.  I would like to thank everyone who replied to this question,
I was grateful for all the suggestions.

I thought it might be useful to summarise the replies for anyone else with
this problem.

Agarase - This involves the incubation of the agarose gel with an enzyme
which digests the agarose, such as Gelase from Epicentre Technologies. 
The problem for me with this is that it requires the removal of the enzyme
if you need to run a gel again, so would require a phenol step. 

Home-made spin columns - This involves punching a hole into a 0.5ml
eppendorf and placing some silicanised glass wool in the bottom, this tube
is then put into a 1.5ml eppendorf tube.  The gel slice is put ontop of
the glass wool and the eppendorf is then spun slowly. 

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%. 

Freeze-fracture of agarose - Place the gel slice at -80 C then spin in a
microfuge, the resulting supernatant should contain your DNA.  An
alterantive to this is to wrap the gel slice in lab film, freeze and then
squash with force.  The resulting liquid will contain the DNA, no
purification should be needed after that,

Extraction using filter paper and syringe - this method was detailed in
BioTechniques 15(6): 976-978 (1993)  Another method is in Lu et al. 1994
BioTechniques 16(3):400-402. 

Again many thanks to those who helped and I hope this mail may help others

Alison Reavley 
MRC Radiation and Genomic Stability Unit
Harwell
UK








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