TITLE: Restriction Digest -- CHEM 384/584
AUTHORS: New England Biolabs, Ken   Christensen

[DESCRIPTION]
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. 

[STEPS]
1. Set up the following reaction (total reaction volume 50 µl).
 
   Restriction Enzyme 10 units is sufficient, generally 1µl is used   DNA 1 µg   10X NEBuffer 5 µl (1X)   Total Reaction Volume 50 µl   Incubation Time 1 hour*   Incubation Temperature Enzyme dependent   
* Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
 
 
 
 
 
 

2. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube. Do not vortex the reaction.
3. Quick ("touch") spin-down in a microcentrifuge.
4. Incubate for 1 hour at the enzyme-specific appropriate temperature. 
60 min