PCR and Agarase

r.nakisa at ic.ac.uk r.nakisa at ic.ac.uk
Mon Mar 8 11:09:13 EST 1993


Dear Netters,

This is to confirm that DNA gel-purified with beta-agarase can't be
amplified by PCR.  I did a control experiment with a template and
primers that ALWAYS work in which I loaded the template onto a LMP gel
and purified it with a beta-agarase digestion.  There was no
amplification.

I have just spent a month trying to amplify my ligation product. 
Because of a sin of omission in the agarase instructions the
amplification was doomed not to work :-(.  The instructions say

   Beta-agarase I acts by cleaving carbohydrate bonds, freeing trapped
   DNA, and producing carbohydrate molecules which can no longer gel. 
   The remaining carbohydrate molecules and Beta-Agarase I will not, in
   general, interfere with subsequent DNA manipulation including:
   restriction endonuclease digestion, ligation and transformation.

And, of course, excluding PCR.

Someone has written to me telling me his lab has found exactly the same
thing with the american version of the enzyme called gelase.  I'm rather
miffed that I wasted so much time for nothing.

Others, beware!

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