There are several key factors to consider when setting up a restriction endonuclease digest. Using the proper amounts of DNA, enzyme and buffer components in the correct reaction volume will allow you to achieve optimal digestion. By definition, 1 unit of restriction enzyme will completely digest 1 µg of substrate DNA in a 50 µl reaction in 60 minutes. This enzyme : DNA : reaction volume ratio can be used as a guide when designing reactions. However, most researchers follow the "typical" reaction conditions listed, where a 5-10 fold overdigestion is recommended to overcome variability in DNA source, quantity and purity. NEB offers the following tips to help you to achieve maximal success in your restriction endonuclease reactions.

A "Typical" Restriction Digest

Most researchers follow the general rules that 10 units of restriction enzyme is sufficient to overcome variability in DNA source, quantity and purity. Generally, 1 µl of enzyme is added to 1 ug of purified DNA in a final volume of 50 µl of the appropriate 1X NEBuffer followed by incubation for 1 hour at the recommended temperature. If an excess of enzyme is used, the length of incubation can often be decreased to save time. Alternatively, you can productively digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.

Enzyme

• Keep on ice when not in the freezer
• Should be the last component added to reaction
• Mix components prior to addition of enzyme by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Follow with a quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
• Supercoiled plasmids and agarose-embedded DNAs generally require more than 1 unit/µg to be cleaved completely.
  

DNA

• Should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents or excessive salts
• Methylation of DNA can inhibit digestion with certain enzymes. For more information about methylation, visit Effect of CpG Methylation on Restriction Enzyme Cleavage and Dam and Dcm Methylases of E.coli.

Reaction Buffer

• Use at a 1X concentration
• If required, add BSA to a final concentration of 100 µg/ml (1:100 dilution)
• Restriction enzymes that do not require BSA for optimal activity are not adversely affected if BSA is present in the reaction
  

Reaction Volume

• A 50 µl reaction volume is recommended for digestion of 1 µg of substrate
• Keep glycerol concentration at less than 5% of total reaction volume to prevent star activity
• The restriction enzyme (supplied in 50% glycerol) should not exceed 10% of the total rxn volume
• Many techniques such as cloning, genotyping, mutational analysis, mapping, probe preparation, sequencing and methylation detection require the use of enzymes under suboptimal conditions.
• Additives in the restriction enzyme storage buffer (e.g., glycerol, salt) as well as contaminants found in the substrate solution (e.g., salt, EDTA, or alcohol) can be problematic in smaller reaction volumes. NEB has introduced a line of High Fidelity (HF) enzymes that provide added flexibility to reaction setup. The following guidelines can be used for techniques that require smaller reaction volumes.
  
  Alternative Volumes for Restriction Digests
  

  	Restriction Enzyme*	DNA	10X BEBuffer	100X BSA** (if needed)
  10 µl rnx***	1 unit	0.1 µg	1 µl	0.1 µl
  25 µl rnx	5 units	0.5 µg	2.5 µl	0.25 µl
  50 µl rnx	10 units	1 µg	5 µl	0.5 µl

  * Restriction Enzymes can be diluted using the recommended diluent buffer when smaller amounts are needed.
  ** BSA can be diluted in 1X buffer.
  *** 10 µl rxns should not be incubated for longer than 1 hour to avoid evaporation.

Incubation Time

• Typically 1 hour
• Can often be decreased by using an excess of enzyme, or by using one of our Time-Saver qualified enzymes.
• It is possible, with many enzymes, to use fewer units and digest for up to 16 hours. For more information, visit Extended Digests with Restriction Endonucleases.

Stopping a Reaction

If no further manipulation of DNA is required:

• Terminate with a stop solution (10 µl per 50 µl rxn) [50% glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue] (e.g., NEB #B7021)

When further manipulation of DNA is required:

• Heat inactivation can be used
• Remove enzyme by using a spin column or phenol/chloroform extraction
  

Storage

• Storage at -20°C is recommended for most restriction enzymes. For a few enzymes, storage at-70°C is recommended for periods longer than 30 days. Please refer to the enzyme's technical data sheet or catalog entry for storage information.
• 10X NEBuffers and concentrated BSA should also be stored at -20°C
• BSA should not be mixed directly into NEBuffers and then frozen because the BSA may precipitate
  

Stability

• All enzymes are assayed for activity every 4 months. The most recent assay date, as well as the expiration date is found on the label
• Exposure to temperatures above -20°C should be minimized whenever possible
  

Control Reactions

If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions:

• Experimental DNA without restriction enzyme to check for contamination in the DNA preparation or reaction buffer
• Control DNA (DNA with multiple known sites for the enzyme, e.g. lambda or adenovirus-2 DNA) with restriction enzyme to test enzyme viability
• If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.

Note: Some enzymes have a high DNA binding affinity and may not dissociate from the product well. This can result in smearing on an agarose gel. In these cases, after the reaction is complete, add SDS to a final concentration of 0.1-0.5%. This may result in a cleaner banding pattern.