Purification from Low Melt
jow at helix.nih.gov
Fri Feb 18 13:02:45 EST 1994
In article <genecutl-170294174345 at kos2mac16.berkeley.edu> gc,
genecutl at mendel.berkeley.edu writes:
>Are there other techniques which give more consistent results?
I'm getting along in years, so my roots go back to the good old days and
carcinogens scare me less since I plan on being dead of other causes
before they get me. Thus, my method involves using phenol and chloroform.
Cut the bands out and put in 1.5ml microfuge tubes. Weigh the agarose in
the tube. (Tubes from the same batch seem to be within 10mg.) Add 450ul
- mg agarose to the tube. Add 50ul 5M NaCl. Melt at 70 deg. Add 0.5ml
phenol (0.1M TrisHCl pH8 saturated), and vortex 30 sec. Spin in
microfuge at maximum for 3-5 min. Remove aqueous (upper) phase to fresh
microfuge tube containing phenol:chloroform [1:1], and vortex 30 sec.
Microfuge for 1-2 min. Remove aqueous (upper) phase to microfuge tube
containing 0.7ml chloroform, and vortex for 30 sec. Spin in microfuge
30-60 sec. Remove chloroform (bottom) phase. Spin another 10 seconds
and remove last few ml of chloroform. Precipitate with 1ml ethanol.
Rinse precipitate with 1ml 70% ethanol. Dry. Dissolve in appropriate
volume of TE.
It works reliably for me.
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