13.4: Lab Technique - Restriction Digest of DNA

Key Components of a Restriction Digest

The key components of a restriction digest are as follows:

• DNA sample: typically a piece of linear DNA or a circular plasmid
  • Amount used in the reaction depends on the application of the DNA (e.g. cloning, sequencing, mapping)
  • Recommended amount for molecular cloning is 1 µg
• Restriction Enzyme: supplied in glycerol so that it will not freeze
  • Sold at a specific concentration that is expressed as "units per microliter" (U/µL)
  • The volume of restriction enzyme used depends on the amount of DNA being digested. One unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour
  • Stored at -20ºC
  • Should be kept on ice until needed
• Restriction Buffer: a concentrated mixture of salts provided with the restriction enzyme
  • Usually provided as a ten-times (10X) concentrated buffer but other concentrations (2X, 5X) are also possible
  • The volume used will depend on the final volume of the restriction digest
  • When diluted to a 1X concentration in the digest, provides an optimal salt concentration and pH for the restriction enzyme
  • Restriction enzymes require the use of restriction buffers with specific compositions. One specific restriction buffer may be suitable for use with several restriction enzymes. However, some restriction enzymes have unique buffers (e.g. EcoRI).
  • When performing a double digest with two enzymes, the buffer with the lower salt concentration is typically used in the reaction to ensure maximal activity of both enzymes. Restriction enzyme activity in several buffers can be verified using the company's webpage (e.g., Double Digest Finder; New England Biolabs)
  • Stored at -20ºC and should be thawed completely prior to use
• 100X BSA (optional): a concentrated mixture of bovine serum albumin
  • May not be required for every restriction enzyme
  • The volume used will depend on the final volume of the restriction digest
  • When diluted to 1X concentration in the digest, provides stability to the restriction enzyme and increases the efficiency of the digest
  • Stored at -20ºC and should be thawed completely prior to use
• Distilled water: used to provide the final volume of the restriction digest
  • The final volume of a restriction digest can vary. Typical final volumes include 20 µL to 50 µL

Lab Protocol: Double Restriction Digest

1. Thaw all frozen components and place on ice along with the desired restriction enzyme(s).
2. Label the desired number of 1.5 mL microfuge tubes appropriately (e.g. "plasmid", "insert").
3. Add the desired amount distilled water to each tube. The amount of distilled water will depend on the final volume of the digest.
4. Add 1.0 µg of the appropriate DNA to each tube (e.g. plasmid DNA, insert DNA).
5. Add the correct volume of restriction buffer to each tube. The exact restriction buffer to be used will be dependent on the restriction enzyme being used. The correct volume will based on the concentration of the buffer and the final volume of the reaction.
6. Optional: Add the correct volume of 100X BSA to each tube based on the final volume of the reaction.
7. Add the correct volume of each restriction enzyme to each tube.
8. Vortex each tube and "flick spin" in a centrifuge to collect the contents at the bottom of each tube.
9. Incubate at 37ºC for 1 hour.
10. Heat inactivate the enzymes by incubating for 10 minutes at 75ºC
11. Store the reaction(s) at -20ºC until needed.