PCR amplication

Protocol 8.

Method

1. To a 0.5 ml microcentrifuge, add 8-10 |xl of the cDNA reaction mixture from above.

2. Add the following reagents. DEP-treated water, 75-77 |xl

10 x reaction buffer (Perkin-Elmer Cetus)/ 10 jjlI

forward primer (25 pmol/ml), 2 |xl reverse primer (25 pmol/ml), 2 jlI

Final concentration is 25 mM TAPS [pH 9.3], 50 mM KCI, 2 mM MgCI2, 1 mM p-mercaptoethanol.

3. Overlay with 50 |xl mineral oil and heat to 94°C for 7 min (alternatively, Taq can be omitted until after the heat denaturation, then pipetted under the mineral oil layer into the aqueous solution).

4. Amplify toc/-2cDNA using the following PCR cycling conditions: Mouse bcl-2: 94 °C, 30 sec 57 °C, 30 sec

72 °C, 3 min (this should be 8 min for the last cycle) Human BCL-2: 94°C for 1 min

72 °C for 1 min, where the 72°C step is lengthened by 8 sec per cycle

(A final extension at 72°C is performed for 10 min after the last cycle) The number of cycles for obtaining results within the linear portion of the reaction varies between cells and tissue types. In general, 25-30 cycles has been appropriate for mouse and 25-20 for human, but a range of cycle numbers should be examined in pilot experiments, e.g. 15, 20, 25, 30, 35 cycles.

The two cycle profiles for mouse and human BCL-2 were independently arrived at by separate workers in the laboratory and have proved to be empirically successful in our hands. No efforts have been made to test the interconvertibility of these cycling profiles for mouse and human BCL-2.

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