MIT iGEM Restriction Digests
Restriction Digest protocol
For Analytical digests, aim for 500-1000ng of DNA; for a preparative digest (where you know you want to cut out a band) try to load as much DNA as possible (gel lanes w/ wide-toothed combs can hold up to 50µl).
- 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.
- BSA (100x from NEB)
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 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
- Add 0.2µl BSA to tube.
- Add 2.0µl 10x Buffer to tube.
- Add appropriate amount of DNA to tube.
- Add 0.5µl of each enzyme to tube.
- MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
- Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
- Deactivate the enzymes by heating for 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
- Keep digest at -20*C or run immediately on gel.