PCR optimizatio

Rafa Maldonado Rafael at genetics.med.utah.edu
Fri Jan 21 23:07:25 EST 1994


HI Doug and everyone
The Tm is calculated by the AT+CG method. There are more complicated
methods, and some programs for calculating more acurately, but in my
hands, the AT+CG method wroks great.
It looks like your agarose have contamination of something! You said
that at higher concentrations of Mg you got lower background. The [Mg]
is necessary for the PCR, but at high concentrations the Mg inhibits
the polymerase.I would say that your agarose has a lot of magnesium,
and increasing the [Mg] you get lower background because your enzyme is
starting to be inhibited. I'm not an expert in Biochemistry, but I
think you should try another make of agarose or some purified grade.
Anyway, life is life, and if you get the backgraound off when
increasing Mg, go ahead!
You can test that desalting some template and see what happens with the
magnesium. I have done PCR with agrarose-extracted DNA by geneclean and
I never had problems.
Unfortunately, the Tanneal has to be tested empirically! Each template
has a diferent sequence, and your primers can have diferent unspecific
annealing with diferent sequences. Anyway, your method of 5 or 6
degrees less than Tm is good, and it works for many cases. I never
change this, unless I want some specific PCR reaction, i.e., very high
yield, or I get background.

See you soon in this screen!



----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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