PCR products from gel

the End jgraham at bronze.ucs.indiana.edu
Sun May 8 11:37:31 EST 1994


Bernard Heymann: >PCR products from gel                   Sat,


Apparently DNA which has once been in agarose is altered in some basic
properties which causes it to be lost in many common manipulations. Therefore,
I find the less you do to it, the higher the yield. Freeze-squeeze based
methods are generally best, however material obtained in this way may be
difficult to precipitate or to purify by organic extraction. If at all
possible, try to use it directly. Residual agarose and EthBr does not
interefere with any enzymatic treatment that I've examined.

As for the size, PCR products <500 bp show great variability in alcohol
precipitations unless the counter-ion is sodium acetate rather than ammonium
acetate (a generaly preferred salt).

Fewer steps, tube transfers, columns, extractions, precipitations, all
increase yield in my exprience. My biggest mistake over the years was trying
to clean up DNA purified in agarose more than necessary for subsequent
manipulations.

If your particualr problem persists, run a 5% acrylamide gel, cut out your
PCR product, grind the gel slice up with a pipette tip, freeze it at -70C,
then soak it several hours or overnight in TE buffer in a shaker at 37C.
The eluted material can be manipulated successfully by common practices
(in contrast to agarose purfied DNA).

Of course, instances of expected behavior from these fragements are also
common (ie. I just precipitated/extracted fine, ect.)  but the variability
involved precludes use as a standard techinique, in my expereince.


Jim
J. Graham
Biology and Chemistry Departments
Indiana University Bloomington
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