Restriction Enzyme Digestions
1. Determine which 1, 2, or 3 restriction enzymes are to be included in the digestion reaction.
Check “NEB double digest finder” to ensure that all enzymes are compatible in the same buffer
type. [Note: most enzymes are being shifted towards “CutSmart Buffer”].
2. Set up reaction in an Eppendorf tube (e.g. 50 uL reaction):
(i) Purified DNA (5-25 uL depending on needs)
(ii) 10x Buffer (5 uL)
(iii) 0.1-0.5 of each enzyme*
(iv) water to final volume of 50 uL
*Keep enzymes in cold trays and only take out of freezer when needed (e.g. final step should be
to add enzymes to reaction tubes). If a large amount of digests are needed, a “Master Mix” of
the Water + buffer can be made and aliquoted to each tube. Do NOT include DNA or enzymes
in the master mix. Ensure that the buffer (frozen) is fully thawed and vortexed to mix prior to
using.
3. Place at 37°C incubator for at least 1 hour. Depending on need (diagnostic, shorter
incubation times; complete digestions for IVLs or subclones, can digest overnight).
4. Add loading dye and run on DNA agarose gel for visualization and/or extraction.