Elution of DNA from agarose gels

Rick Wilson rick at GENEMAN.WUSTL.EDU
Wed Nov 13 10:44:40 EST 1991


K.R. Raj writes:
>I am quite tired of electroeluting DNA out of agarose. Could
>anyone suggest a better way other than using the geneclean kit ?
>                  Thankyou.
>                                               kraj at uk.ac.crc

I've had a lot of success with simple phenol extraction of melted
low-gelling temperature agarose.  Cut out the bands, trim away excess
agarose, place the gel slice in a 1.5 ml microfuge tube.  Place at
65 degrees for 5 minutes.  Bring volume to 0.5 ml with TE buffer.  Add
an equal volume of Tris-saturated (pH 8) phenol, vortex vigorously,
place at -70 degrees for at least 15 minutes (or on dry ice for at
least 5 minutes).  Remove tube from freezer (or dry ice) and place
directly in a microfuge.  Spin at top speed for 5 minutes.  Remove the
aqueous (top) phase to another microfuge tube.  Extract twice with
one-half volume of phenol, and once with one volume of water-saturated
ether.  Add one-tenth volume of 3M NaOAc, pH 5.2 and two volumes of
ethanol.  Place at -70 degrees for at least 15 minutes.  Centrifuge at
room temperature for 15 minutes to pellet DNA.  Wash once with 1 ml of
70 percent ethanol and dry briefly under vacuum.

In my hands, recovery of 85-95 percent is typical.  In most cases, I
use the eluted DNA for cloning or direct sequencing with no further
purification.

R. Wilson
St. Louis




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