Assemble reaction mix into 50 µL volume in a microfuge tube.
Add reagents in following order: water, buffer, BSA, DNA template, restriction enzyme.
Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
Prepare negative control reaction without template DNA.
Prepare positive control reaction with template of known cutting site corresponding to the restriction
enzyme of choice.
Typical Incubation time and temperature is 37°C for 1 hour, though time and temperature will vary
depending on restriction enzyme used.
Restriction enzymes are typically inactivated by incubation at high temperature. Incubation time and
temperature is 65°C for 20 min, though time and temperature will vary depending on restriction enzyme
Analyze the results of your PCR reaction via gel electrophoresis.
to 50 µL
5-10 U per µg of DNA template (should not exceed 10% of reaction volume)