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Time: 20 min

Reference: NEB.com


  • (optional) Determine plasmid concentration via A260 measurements or by comparing intensities of bands on gel with those of the ladder DNA (whose masses are known).


  1. To an eppendorf tube, add:
    1. Appropriate volume of plasmid for a total of approx. 700 ng DNA (usually, if plasmid prep is good, this will be 2 μL (i.e. ~350 ng/ μL)).
    2. 1 μL of appropriate 10X concentration buffer (to determine correct buffer check compatibility of restriction buffers using NEB catalogue or going online to NEB.com [1])
    3. If necessary (i.e. check NEB), add 1 μL 10X concentration BSA
      • BSA~bovine serum albumin
    4. [x] μL of dH2O to make the total reaction volume in tube of 10 μL.
    5. Chosen restriction enzymes. They are at a high enough concentration that 0.5 μL of each is more than sufficient for a restriction digest. ALWAYS ADD THESE LAST (and work quickly), in order to minimize time out of the freezer. Keep these enzymes in their low-temp blue carrying case when out of the freezer.
  2. (Gingerly) flick tubes, and spin down in microcentrifuge for a second
  3. Incubate at 37°C (in water bath or warm room) for 1-4 hrs. (1-2 hrs. optimal)
  4. (Under review)...treat any vectors w/0.2 μL CIP and incubate for 30 min at 37°C with rest of restrictions

Suggested NEBuffers for Double Digestion
Suggested NEBuffers for Double Digestion




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