To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube:
- 1000 ng of DNA or up to 20.5 ul of DNA (if performing the excision of an insert for a ligation, e.g. E-P, X-P, E-S) or
- 500-700 ng of DNA or up to 20.5 ul of DNA (if opening a plasmid E-X or S-P for a ligation) or
- 50-1000 ng of DNA (200 ng is ok for most cases) or up to 20.5 ul of DNA (if performing a screening)
- Milli-Q water up to 20.5 ul.
- 2.5 ul of buffer H
- 1 ul of the first enzyme
- 1 ul of the second enzyme
Mix very well and incubate in the thermocycler (Digestion
program, reaction volume of 25 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.