John M. S. Bartlett.pdf - Bio-Nica.info

Site-Directed Mutation and PCR Mimics 207
Fig. 1. Example of a crossover plot for a sample containing 10 pg of RI alpha cDNA. Vertical
axis, counts per minute. Horizontal axis concentration of RI alpha mutant cDNA added. Solid
curve = mutant RI alpha plasmid, dashed curve = sample. Reproduced with permission from
Bartlett et al. (1).
3. Prepare PCR master mix as follows: per tube, add To each tube 5 µL of 10× reaction
buffer, 5 µL of 25 mM MgCl 2 , 0.1 µL of Taq polymerase, 5 µL of radioactive dNTP mix,
and 14.9 µL of distilled water.
4. Add 30 µL of PCR master mix to each tube set up in steps 1 and 2. If not using a heated
lid PCR block, overlay with 100 µL of paraffin oil.
5. Perform PCR amplification for 26 cycles (94°C for 40 s, 55°C for 60 s, 72°C for 70 s)
followed by extension at 72°C for 5 min.
3.3. Restriction Digestion
1. Add 5.0 µL of 100 mM sodium chloride to each reaction followed by 1.0 µL of EcoRV
restriction enzyme (10 units; see Note 10).
2. Incubate at 37°C for 2 h to ensure complete digestion of mutant RIα product.
3. Labeled PCR products were separated on a 6% polyacrylamide gel at 30 mA for 2 to 3 h
using a Protean II vertical electrophoresis system (Bio-Rad UK).
4. Gels were fixed for 30 to 60 min in fixative and dried using a flat bed gel dryer and heating
to 80°C for 1 to 2 h.
5. Gels were exposed to preflashed X-OMAT (Kodak UK) film for 1 to 8 h using radioactive
ink to orient the gels.
6. Bands corresponding to normal (430 base pair) and mutant component (215 base pair)
component of each reaction were excised and 32 P incorporation determined by Cerenkov
counting (see Note 11).