Endy:Restriction Digest

Materials

 * Prepped DNA. For a BioBrick assembly (and possibly any cloning operation) 300 ng is more than enough.


 * Restriction enzymes (EcoR I, Spe I, Xba I or Pst I)


 * Restriction buffers (also from New England Biolabs. source)
 * For all combinations of the four BioBrick enzymes, we use Buffer 2 + BSA.
 * ddH2O

Digest Mix

 * 0.2-1&mu;g DNA from prep or PCR
 * 10% (by volume) Restriction Buffer
 * 1% (by volume) BSA stock
 * 2.5% - 5% (by volume) of each restriction enzyme.
 * ddH2O to produce correct percentages by volume
 * Enzymes < 10% of total volume so glycerol is < 5% of total volume.

We prepare a restriction digest supermix (stored at -20C) containing - where n is the desired number of 50&mu;l reactions.
 * 5*n &mu;l of Buffer 2
 * 0.5*n &mu;l of BSA
 * 37*n &mu;l of ddH2O

Example - 50&mu;l digest

 * 42.5&mu;l of restriction digest supermix
 * 1-5&mu;l of preppedDNA
 * 1&mu;l of each restriction enzyme

Reaction conditions

 * Incubate reaction at 37&deg;C for at least 1 hour; longer digest gives more complete digestion, especially if you have >1µg DNA, but can at times give nonspecific digestion, even if glycerol is <5%. Heat kill enzymes at 80C for 20 min.
 * Store at -20&deg;C.