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).

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)

Method
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)
 * 1) Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
 * 2) Add 0.2µl BSA to tube.
 * 3) Add 2.0µl 10x Buffer to tube.
 * 4) Add appropriate amount of DNA to tube.
 * 5) Add 0.5µl of each enzyme to tube.
 * 6) MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
 * 7) Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
 * 8) Deactivate the enzymes by heating @ 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.
 * 9) Store digest at -20*C or run immediately on gel.