Restriction Digest -- CHEM 384/584
Ken Christensen, New England Biolabs
Published: 2022-02-01 DOI: 10.17504/protocols.io.b4hvqt66
Abstract
The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.
Before start
Steps
1.
Enzyme volume should not exceed 10% of the total reaction volume to prevent Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol. due to excess glycerol. A 50 µl reaction volume is recommended for digestion of 1 µg of substrate. The enzyme should be the last component added to reaction Keep Enzyme in the enzyme storage box while at the bench rather than removing it and placing it on ice.
Set up the following reaction (total reaction volume 50 µl ).
| A | B |
|---|---|
| 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 |
-
Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
Note
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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. Can be decreased to 5-15 minutes by using a Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.. See the See the NEB Activity/Performance Chart for the incubation temperatures. for the incubation temperatures.
Incubate for 1 hour at the enzyme-specific appropriate temperature.
1h 0m 0s
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