EcoRI/SpeI Double Digest

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I usually use the supplied EcoRI buffer for EcoRI/SpeI double digests, as recommended in your catalog. However, I always find that this digest is quite inefficient, and I noticed in your current catalog that you report an activity of 25% for SpeI in the EcoRI buffer.

Would you recommend doing this digest in Buffer 2, the recommended buffer for SpeI, since EcoRI has 100% activity in Buffer 2? Would you expect this to cause problems with star activity?

Answer (from NEB technical support):

Yes, "Star Activity" is the brief answer.

A longer answer: EcoRI has full activity in many reaction buffers, but its specificity is poor in almost all of them. The sole reason which promted us to create the unique reaction buffer for EcoRI is avoidance of Star Activity. [Star Activity is defined as any endonucleic digestion at sites which resemble the recognition site, but are not identical to it.]

If you were to limit the duration of the digestion, and limit the ratio of EcoRI to DNA, and limit the glycerol concentration, then EcoRI Star Activity is avoidable on many substrates when using some alternative reaction buffers. But, on difficult substrates (i.e., those which require an excess of EcoRI or prolonged digestion, such as impure DNA, genomic DNA or any agarose-embedded DNA) complete digestion by EcoRI many not be attainable without also causing Star Activity, when used in NEBuffer 1, NEBuffer 2, or NEBuffer 4.

SpeI and EcoRI The rate of digestion of by SpeI in NEBuffer EcoRI is 25%. This is the lower limit that we choose as acceptable for recommended use.

If you wish maximal efficiency, and if the substrate is not a difficult one to cut, then a double digestion by SpeI and EcoRI can be performed in 1X NEBuffer 2 +BSA provided that the following limits are adhered to:

  1. no more than 10 units of EcoRI per microgram of DNA
  2. one hour incubation at 37C (not longer)
  3. limiting glycerol to 2.5%.
    • This requires using a reaction volume of 20 times the combined volume of the two enzymes (e.g., using 0.5 ul of EcoRI and 0.5 ul of SpeI requires a minimum reaction volume of 20 ul.)

Feel free to e-mail to <> if you have further questions or comments.