Sequential Double Digest
Larissa de Clauser
Abstract
This is the Sequential Double Digest Protocol with Standard Restriction Enzymes. If there is no buffer in which the two enzymes exhibit > 50% activity, this sequential digest can be performed.
Before start
NEB's online tools, Double Digest Finder and NEBcloner will help guide your reaction buffer selection when setting up double digests.
Steps
Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (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.
Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease.
Add the second enzyme.
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.
