PCR restriction cut

Bernhard Mayr ndxdbmay at rrzn-user.uni-hannover.de
Thu Apr 11 16:28:39 EST 1996


I've recently had a problem cutting my PCRs with AluI.

What turned out to be the problem was detergent.

I substituted the supplied  buffer with 20mM TrisCl, 50 mM KCl pH8.4 (1x)
for PCR and adjusted MgCl to 10mM after PCR before cutting with AluI and
it worked. But strikingly not for all Taqs from different
suppliers: One Taq I use has ~0.1% Tween 20 in its storage buffer and
obviously even a 1:250 dilution (0.2 µl Taq in 50µl PCR) is to much for
AluI because when using this Taq I got only partial digestion (everything
else wasw identical).

So maybe using another buffer or Taq might solve the problem.

Bernhard Mayr
Dept Clin Endocrinology
Medizinische Hochschule Hannover, Germany




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