Ammonium in PCR buffers

Robert Preston rapr at MED.PITT.EDU
Wed Jul 27 19:04:57 EST 1994


Hi, netfolks:
I've gotten myself into LA-PCR a la Wayne Barnes, and notice that the
recommended buffer has 16mM (NH4)2SO4 (i.e., 32 mM NH4) in it.  Also,
Barnes advocated high pH (>9) for LA-PCR.  So, with all the talk about
35S derivatives leaking through PCR tubes and contaminating peoples' cyclers,
I wondered whether or not loss of volatile NH3 via permeation through thin-
wall cycler tubes might throw the pH toward acid, thereby de-optimizing
the LA-PCR system.  I tested a 16mM (NH4)2SO4 + 50 mM TrisHCl buffer,
initially at pH 9.0, to see how large a change might happen, in a worst case.
After brief nuking, the pH had dropped to 8.4!! That change, coupled with the
drop in Tris pH by 1.5 units on going from 25deg to 95 deg (due to temperature-
dependence of the pKa of Tris), would put a PCR reaction at pH 6.9, maybe a
bit too acidic for LA-PCR.  However, when I heated the buffer in Perkin-Elmer
MicroAmp tubes (0.2ml) in a P-E 9600 at 94degrees for an hour and a half,
the pH dropped from 9.0 to 8.9, indicating very low loss of NH3 from those
tubes.  So it looks like this isn't a problem in my system.  I bring it up
for discussion because other plastics, or oiled operations, might not be so
immune to loss of NH3 and the resulting pH drop.

Rob Preston
rapr at med.pitt.edu




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