Sequential Double Digest
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 .
