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 .
