Restriction Digest

Protocol

1. Check the NEB (or other supplier) website to confirm enzyme compatibility
2. In a PCR tube add the reaction components according to the table listed below
3. Mix reaction gently and spin down briefly with benchtop centrifuge
4. Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not

Example Reaction

Component	Volume
ddH2O	Up to 50 µL
10X rCutSmart Buffer	5 µL
DNA	x (1 µg)
Enzyme 1	1 µL
Enzyme 2	1 µL

Notes

- NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified

- NEB has the option to select your specific enzymes and generate a detailed protocol - Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step

References, related resources, and acknowledgments

https://nebcloner.neb.com/#!/redigest