Mouse Radiation Hybrid Mapping Project PCR conditions

Typical PCR conditions are as follows :


        21 ng           DNA
        1.25 nmoles     dNTPs
        15 pmoles       of each primer
        1 X             PCR buffer  (20 mM  Mg2+)
        1.4 µl          loading buffer
        0.5 U           Promega Taq polymerase


The final reaction volume is 10 µl. 
The thermal cycling is done on a "waffle iron" (for 16x192 microtiter plates)
using the following conditions with hot start:


        96 C              5 min
        94 C            15 min

        94 C            20 sec
        61 C            30 sec  x 3 cycles

        94 C            20 sec
        59 C            30 sec  x 3 cycles

        94 C            20 sec
        57 C            30 sec  x 3 cycles

        94 C            20 sec
        55 C            30 sec
        72 C            10 sec  x 29 cycles

        72 C            60 sec

The amplified product is analysed on agarose gels (3% Nusieve, 1% Seakem)
and, after ethimium bromide staining.
Electrophoresis is in 1X TBE for 1 hour at 180 Volts.