Freeze & Squeeze: DNA from Low-melt

Bruce Roe BROE at AARDVARK.UCS.UOKNOR.EDU
Thu Oct 24 07:40:00 EST 1991


 You write: 
=> 
=> I have a collegue in need of a recipe [ it seems he left home without his book
=> of protocols].
=> 
=> He has described to me a technique which he calls freeze & squeeze, for 
=> isolating DNA from low-melt agarose.  The excized band is diluted in buffer 1
=> [this is where the cookbook is important!], frozen with liq N2, and centrifuged
=> through a tiny hole in the bottom of a 0.5ml eppendorf tube (into a 1.5ml tube).
=> 
=> I know a number of ways to isolate DNA from agarose, but this one intrigues me....  does anyone out there have the complete protocol??
=> 
=> thanx in advance.
=> 
=> Nigel Walker        nigel at enzyme.berkeley.edu       (510) 654-LEAF

Nigel,
	The buffer you refer to could be:

"oligo GEB": from  Meth. Enz. 65, 506.

	500mM  NH4OAc	1 ml of 5M stock
	10mM  MgOAc	0.1 ml of 1M stock
	1mM  EDTA	0.02 ml of 0.5M stock
	ddH2O	to 10 ml

Some folks also add a small amount of SDS to this buffer and say it helps.
I have tried the protocol you mention and there are commercially available
spin columns for this but if you use the pin-hole, you may wish to add some
siliconized glass wool to prevent the gel from being extruded through the
hole.

We use the following:

Elution of DNA from agarose gels
 
1.  The DNA sample is loaded onto a low-melting preparative agarose gel (0.7 to
1.0%), and electrophoresed as required.  Ethidium bromide (20 5l of 10 mg./ml)
may be included in the gel.
 
2.  Visualize DNA bands on a long wave UV glow box and photograph.  Excise the
desired DNA bands with a clean, ethanol-washed scalpel.  Transfer the gel slice
to a clean microfuge tube.
 
3.  Melt the agarose at 65 deg C for 5 minutes.  Add one volume of TE-saturated
phenol and vortex for a few seconds.  (Optional:  centrifuge for 3 minutes and
then skip to step 6.)

4.  Freeze the agarose-phenol mixture at -70 deg  C for at least 15 minutes.
 
5.  Thaw the sample for a few minutes at room temperature.  Centrifuge for 3
minutes.

6. Remove the aqueous phase to a clean microfuge tube.  Add one-half volume of
phenol, vortex and centrifuge for 2 minutes.  Repeat phenol extraction one more
time.

7.  If sample volume is 0.5 ml or less, skip to step 8.  If sample volume is
greater than 0.5 ml, add 1.5 volumes of n-butanol, vortex and centrifuge for 2
minutes.  Remove butanol (upper) phase and discard.  Butanol extraction may be
repeated until the sample volume is 0.5 ml or less.

8. Extract once with one volume of water-saturated diethyl ether.  Centrifuge
for 1 minute to separate phases, discard ether (upper) phase.

9.  Add one-tenth volume of 3M NaOAc, pH 5.2 and 2 volumes of cold 95% ethanol.
Precipitate DNA by storing at -70 deg C for at least one hour.

10.  Pellet the DNA by centrifugation for 15 minutes at 4 deg C.  Wash once with
0.5 ml of 70% ethanol and dry for a few minutes under vacuum.

Cheers...............bruce

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