Purifying DNA from gel slices

Mayumi Yagi myagi at u.washington.edu
Fri Nov 8 12:33:42 EST 1996


On Fri, 8 Nov 1996, Alison Reavley wrote:

> 
> Hi, I'm looking for a method of purifying plasmid DNA from gel slices
> which does not involve phenol or any increase in temperature.  At the
> moment I am trying electroelution but find that I am precipitating agarose
> as well as DNA after the elution.  I need to get rid of this agarose again
> without phenol or heating. I have also tried using Quiagen Qiaquick Gel
> Extraction columns but have been frustrated by the small yield and I am
> now using concentrations of DNA above those recommended in the protocol. 
> 
> Any suggestions?
> 
> Alison Reavley
> MRC Radiation and Genome Stability Unit,
> Harwell,
> Didcot UK.
> 
> 
> 
Hi, Alison,

I have an ancient procedure I used when I was in grad school.
Hydroxlapatite purification of fragments from agarose
1.  Run agarose gel in TAE buffer and cut out fragments as usual.
2.  Pour gel for elution:  Embed the agarose gel slices in another gel,
lengthwise directly in front of a tooth in the gel comb, and set slightly
higher than the bottom of the well that will be formed.  I do this by
pouring a thin layer of agarose that just touches the bottom of the gel
comb, letting it harden until just set, then carefully lining up the gel
slice and pouring more hot agarose to fill the gel mold.
3.  When the second gel is fully hardened, fill the well with
hydroylapatite (Prep:  Shake dry powder into TAE in a bottle; autoclave).
4.  Electroelute the DNA into the HAP.
5.  Meanwhile, prepare a column.  I used to use a siliconized glass
Pasteur pipet plugged with glass wool; I think the disposable minicolumns
they sell for spin columns should work as well.  In the bottom layer about
0.2 ml DOWEX AG50W-X8 (Anion-exchange resin), fill the column with 1.5-2ml
G-50 equilibrated with TE. (Prep the DOWEX by washing in 1N NaOH until the
pH reaches 8; rinse and store in sterile 0.5M NaCl/0.1M Tris, ph 7.4).
6.  Load the HAP on top of the packed column, rinsing out the well to get
all of the HAP transferred.
7.  Elute the DNA from the HAP:  after the last of the buffer has drained
down and the HAP is layered more or less evenly on top of the G50, start
the elution: Twice with 250 microliters 1M Na PO4, pH 6.5, then three
times with 300 microliters TE.  Collect each fraction into a separate
microfuge tube.
8.  Check all fractions for your DNA.  Usually it is in fractions 3 and 4,
with some trailing into 2 and 5 on occasion.

If you need more info, e-mail me.  Hope this helps.

------------------------------------------------------------------------------
Mayumi Yagi, Ph.D.
Research (151)
Veterans Affairs Medical Center
1660 South Columbian Way
Seattle, WA 98108
Tel (206)762-1010, ext 1918
Fax (206)764-2598





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