Multi-temperature Double Digest (single buffer)
Set up the following reaction with Enzyme #1 (total reaction volume 50 µl).
Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
Quick ("touch") spin-down in a microcentrifuge.
Do not vortex the reaction.
Incubate for 1 hour at the enzyme-specific appropriate temperature.
Add the second enzyme.
Incubate for 1 hour at the enzyme-specific appropriate temperature.
