USTC/Notebook/Restriction Endonuleases Double Digestion

< Back to Notebook

Restriction Endonuleases Double Digestion
Materials
 * DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
 * Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
 * Appropriate enzymes.
 * ddH2O
 * BSA (100x from NEB)

Procedure

The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer, etc)

add the following components to PCR tube: eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH2O)
 * 2µl BSA (diluted to 10X beforehand)
 * 2µl 10x Buffer
 * appropriate amount of DNA
 * 0.5µl of each enzyme
 * appropriate amount of ddH2O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl
 * MIX the reaction by stiring
 * Incubate the reaction at 37*C for 2-4hrs to ensure complete digestion.
 * Store digest at -20*C or run immediately on gel.