Optimal conditions for PCR of small inserts

Richard Vogt vogt
Tue Jul 25 10:49:55 EST 1995


The size is not a problem for optimization; only for visualization.  Run your
reactions with a spike of 32PdATP (old stuff no one wants works), separate by
8% PAGE (Maniatis) and expose to film.  You can use the dyes to approximate
migration position (Maniatis again).

The degenerate primer situation is another matter.  I have an old Cetus I run
these on.  I have always had great luck with the following profile

92C	3 min

92C	30 sec
37C	1 min
ramp to 72	2 min
72	2 min
5 cycles

92C	30 sec
47C	1 min
ramp to 72	2 min
72	2 min
30 cycles

end or 72 and end

Another machine will need different profiles but the main deal seems to be low
temps and long ramps.

Good luck!




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