Optimizing Restriction Endonuclease Reactions
A "Typical" Restriction Digestion
|Restriction Enzyme||10 units is sufficient, generally 1µl is used|
|10X NEBuffer||5 µl (1X)|
|Total Reaction Volume||50 µl|
|Incubation Time||1 hour*|
|Incubation Temperature||Enzyme dependent|
- Keep on ice when not in the freezer
- Should be the last component added to reaction
- Mix components 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.
- In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.
- NEB has introduced a line of High-Fidelity (HF™) enzymes that provide added flexibility to reaction setup.
- Should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents or excessive salts. Extra wash steps during purification are recommended.
- 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.
- Use at a 1X concentration
- A 50 µl reaction volume is recommended for digestion of 1 µg of substrate
- Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol
- 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. The following guidelines can be used for techniques that require smaller reaction volumes.
Alternative Volumes for Restriction Digests
Restriction Enzyme* DNA 10X NEBuffer 10 µl rxn*** 1 unit 0.1 µg 1 µl 25 µl rxn 5 units 0.5 µg 2.5 µl 50 µl rxn 10 units 1 µg 5 µl
*** 10 µl rxn should not be incubated for longer than 1 hour to avoid evaporation.
- Incubation time is typically 1 hour
- Can often be decreased by using an excess of enzyme
- Can be decreased to 5-15 mins 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 ReactionIf no further manipulation of DNA is required:
- Terminate with a stop solution (10 µl per 50 µl rxn) [2.5% Ficoll®-400, 11 mM EDTA (pH 8.0), 3.3 mM Tris-HCl, 0.017% SDS, 0.015% bromophenol blue] (i.e., NEB #B7021)
- Heat inactivation can be used
- Remove enzyme by using a spin column or phenol/chloroform extraction
- 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 product page for storage information.
- 10X NEBuffers should also be stored at -20°C
- All enzymes are assayed for activity every 4 months. The expiration date is found on the label.
- Exposure to temperatures above -20°C should be minimized whenever possible
Control ReactionsIf you are having difficulty cleaving your DNA substrate, we recommend the following control reactions:
- 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.