Mar 02, 2018

Public workspaceRestriction Digest V.2

DOI
dx.doi.org/10.17504/protocols.io.isycefw
  • New England Biolabs1
  • 1NEB, Ipswich, MA
  • New England Biolabs (NEB)
  • Molecular Biology
New England Biolabs
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DOI: dx.doi.org/10.17504/protocols.io.isycefw
External link: https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
Protocol Citation: New England Biolabs 2018. Restriction Digest. protocols.io https://dx.doi.org/10.17504/protocols.io.isycefw
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
Created: July 05, 2017
Last Modified: August 18, 2020
Protocol Integer ID: 6712
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 If you are using a Master Mix, see 'optimizing RE-Mix® reactions'. 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
Restriction Enzyme10 units is sufficient, generally 1µl is used
DNA1 µg
10X NEBuffer5 µl (1X)
Total Reaction Volume50 µl
Incubation Time1 hour*
Incubation TemperatureEnzyme dependent
* Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme. Enzyme
  • 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. 
DNA
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*DNA10X NEBuffer
10 µl rxn**1 unit0.1 µg1 µl
25 µl rxn5 units0.5 µg2.5 µl
50 µl rxn10 units1 µg5 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
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 NEBuffers 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 50µl).
Restriction Enzyme10 units is sufficient, generally 1µl is used
DNA1 µg
10X NEBuffer5 µl (1X)
Total Reaction Volume50 µl
Incubation Time1 hour*
Incubation TemperatureEnzyme dependent
* Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
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 on ice when not in freezer.
Protocol
Restriction Digest Reaction
NAME
Restriction Digest Reaction
CREATED BY
New England Biolabs
DNA 1µg
Amount1 µg
10X NEBuffer 5µl (1X)
Amount5 µL
Restriction Enzyme,  10 units is sufficient, generally 1µl is used
Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
Incubate for 1 hour at the enzyme-specific appropriate temperature.
Duration01:00:00
Note
Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
Note
See the NEB Activity/Performance Chart for the incubation temperatures.