Purification from Low Melt
jgraham at bronze.ucs.indiana.edu
Sun Feb 20 10:50:53 EST 1994
You mention that you are trying the direct "slice-melt-freeze-spin" process
that is mentioned at the end of the FAQ ? If you are seeing variability
with this method, in which the visualized band is placed in a microfuge
tube, not extracted, percipitated, or otherwise given an opportunity
to "disappear", then you must have a difference in low-melt agarose
or gel buffer (TAE).
Athough a certain amount of DNA fragment is removed with the disrupted
agarose pellet (usually an amount equal to that found in an equal sized
aliquot of the supernatant), this is not ordinarily a problem as you still
have a concentrated solution of your fragment about 200-300 ul from an
average gel slice.
You aren't trying to precipitate from the "slice-melt-freeze-spin"
supernatant are you ? This material ligates with only a 2-3X fold reduced
efficiency and is suitable for restiction and T4 pol end-filling as well.
I have yet to find a reaction for which it is unsuitable, residual
agarose (FMC "Seaplaque"), EtBr, TAE and all.
Biology and Chemistry
Indiana University Bloomington
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