The PCR settings we use to amplify the DNA are:
Initial denaturing at 95°C for 5 minutes
35 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds
Final extension at 72°C 10 min.
We isolated the ACTn3 gene by digesting it with DdeI. This enzyme cuts the strand of DNA at just the right base pair which gives us the strand we're looking for. Once we have the ACTN3 gene isolated, we can run gel electrophoresis and compare it to a DNA ladder to see if we have strands of the right length and then to see which allele each sample contains. If all goes well, the R577X fragment should be 108 base pairs and the R577 fragment should be 205 base pairs.