Restriction Enzyme Tips

Here are some tips for improving your restriction enzyme digestions. Additional optimization recommendations are available.

  1. Enzymes that have low activity in salt-containing buffers (NEBuffer 3.1 or NEBuffer r3.1) may be salt-sensitive. DNA purification procedures that use spin columns can result in high salt levels, which can inhibit enzyme activity. To prevent this, the DNA solution volume added to the reaction for these enzymes should be no more than 25% of total reaction volume.
  2. Cleanup of the PCR fragment (e.g., Monarch® PCR & DNA Cleanup Kit) prior to restriction digestion is recommended. PCR components can inhibit enzyme activity. In addition, the polymerase present in the PCR is active during the digestion step, and can modify the newly created ends by blunting them.
  3. Some enzymes may bind tightly to the substrate DNA. This binding can result in smearing or the presence of unexpectedly high molecular weight bands on a gel. To prevent this, add SDS to a final concentration of approximately 0.1%, or use Gel Loading Dye, Purple (6x), which contains sufficient SDS to dissociate the enzyme from the substrate.
  4. To prevent star activity, make sure that you use the recommended buffer, that the amount of glycerol in the reaction is no more than 5% of the total reaction volume, and that you don’t use too many units or let the reaction go for too long (several hours to overnight). Time-SaverTM qualified enzymes have a lower star activity and are a great choice to prevent this from happening.
  5. Time-Saver qualified enzymes can cut substrate DNA in 5-15 minutes and safely digest overnight. For enzymes that are not Time-Saver Qualified, the recommended incubation time is 1 hr. In general, long incubations (several hours to overnight) are not recommended, unless digesting some gDNAs.
  6. If the restriction enzyme(s) used can be heat-inactivated, then we recommend a heat-inactivation step. This is particularly important if you are planning to move on to the next step in the cloning workflow without a cleanup step. This prevents many different DNA-binding proteins from being present in the same reaction, competing for the same substrate.
  7. If your digestion results in several DNA fragments, and you are only interested in using one of them in your cloning workflow, then the most common way to isolate the fragment of interest is by running the digestion in a gel, excising the band from the gel, and isolating the DNA by column purification (e.g., Monarch® DNA Gel Extraction Kit).
  8. Some enzymes cannot cut the recognition site if the sequence is methylated. Make sure that you check the methylation sensitivity of the enzyme that you are working with. If an enzyme is sensitive to bacterial methylation you may need to grow the substrate DNA in a methylation-deficient bacterial strain.
  9. Some enzymes require two sites for optimal digestion. When a single recognition site is present in a sequence, the second site can be added to the reaction in the form of a short double-stranded oligo containing one, or several, copies of the enzyme’s recognition site.

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at