Restriction Enzyme Digest Protocol

Find more protocols and selection guides in the Molecular Biology Guide.


Sigma® Life Science’s restriction enzyme collection has been optimized for digestion using five unique buffers. When digesting DNA using a single enzyme, use the buffer supplied with the enzyme (also identified on table 1 of the Restriction Enzyme Buffer Reference).

Protocol for DNA Digestion with a single restriction enzyme

  1. Add components to a clean tube in the order shown:
         1 µL DNA (concentration 1 µg/µL)
         2 µL 10x buffer
         1 µL restriction enzyme
         16 µL sterile water
  2. Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
  3. Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
  4. The digested DNA is ready for use in research applications.

When using two restriction enzymes at once, first check the enzyme activities in each buffer, using the table on the Restriction Enzyme Buffer Reference. If they both have 100% activity in the same buffer, you can proceed with your double digestion protocol using that buffer. Alternatively, the optimal buffer can be determined from the chart of common double digestions. In some cases, sequential digestion is recommended due to buffer incompatibility (composition or temperature).

Protocol for DNA Digestion with two restriction enzymes

  1. Add components to a clean tube in the order shown:
         1 µL DNA (concentration 1 µg/µL)
         2 µL 10x buffer
         1 µL each restriction enzyme
         15 µL sterile water
  2. Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
  3. Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
  4. The digested DNA is ready for use in research applications.

Protocol for sequential DNA Digestion

  1. Add components to a clean tube in the order shown:
         1 µL DNA (concentration 1 µg/µL)
         2 µL 10x buffer
         1 µL restriction enzyme
         16 µL sterile water
  2. Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
  3. Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final concentration EDTA.
  4. Recover the DNA using a purification kit: re-suspend and dilute the DNA to 1 µg/ µL.
  5. Prepare second digestion according to step 1. Continue through step 3.
  6. The digested DNA is ready for use in research applications.