Jan 31, 2022
Restriction Digest -- CHEM 384/584
Forked from Restriction Digest -- CHEM 584
- New England Biolabs1,
- Ken Christensen2
- 1NEB, Ipswich, MA;
- 2Brigham Young University
External link: https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
Protocol Citation: New England Biolabs, Ken Christensen 2022. Restriction Digest -- CHEM 384/584. protocols.io https://dx.doi.org/10.17504/protocols.io.b4hvqt66
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 31, 2022
Last Modified: January 31, 2022
Protocol Integer ID: 57621
Abstract
The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.
Guidelines
Guidelines for Optimizing Restriction Endonuclease Reactions
Enzyme
- Keep in the enzyme storage box (Cool Box) when not in the freezer . Try to not remove the enzyme tube from the box while pipetting. Aliquots of enzymes may be provided during class where many students are digesting DNA simultaneously.
- 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.
- High-Fidelity (HF®) enzymes provide added flexibility to reaction setup.
DNA
- 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, Effect of CpG Methylation on Restriction Enzyme Cleavage and Dam and Dcm Methylases of E.coli
Buffer
- Use at a 1X concentration
- Supplement with SAM (S-Adenosyl methionine) to the recommended concentration if required.
Reaction Volume
- 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.
| 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 |
* Restriction Enzymes can be diluted using the recommended diluent buffer when smaller amounts are needed.
** 10 µl rxns should not be incubated for longer than 1 hour to avoid evaporation.
Incubation Time
- Incubation time is 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) [1x: 2.5% Ficoll®-400, 10mM EDTA, 3.3mM Tris-Hcl, 0.08% SDS, 0.02% Dye 1, 0.001% Dye 2, pH 8.0@25°C] (e.g., NEB #B7024 )
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 CutSmart Buffer should also be stored at -20°C
Stability
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 Reactions
If 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.
Set up the following reaction (total reaction volume 50 µl).
| Restriction Enzyme | 10 units is sufficient, generally 1µl is used | |
| DNA | 1 µg | |
| 10X CutSmart Buffer | 5 µl (1X) | |
| Total Reaction Volume | 50 µl | |
| Incubation Time | 1 hour* | |
| Incubation Temperature | Enzyme dependent |
Note
Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
Note
A 50 µl reaction volume is recommended for digestion of 1 µg of substrate.
Note
The enzyme should be the last component added to reaction
Note
Keep Enzyme in the enzyme storage box while at the bench rather than removing it and placing it on ice.
Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Do not vortex the reaction.
Quick ("touch") spin-down in a microcentrifuge.
Incubate for 1 hour at the enzyme-specific appropriate temperature.
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