Single-temperature Double Digest
Updated 2/8/2020 07:36 PM
Introduction
Materials
- DNA 1µg
- 1X
- Deionized Water
- waterbath or whatever is used for incubation & make sure you set it up at the right temperature prior starting the DD reaction
Procedure
Single Temperature DD Reaction
- Set up the following reaction (total reaction volume 50µL).
Table2
A A | B B | C C | |
1 1 | Reagent Volumes (µl) | ||
2 2 | Buffer (10x) | 5 | |
3 3 | DNA * | Input Volume for ng | |
4 4 | Restriction Enzyme #1 ** | 1 | |
5 5 | Restriction Enzyme #2 ** | 1 | |
6 6 | Nuclease free water | B7-B5-B4-B3-B2 | Check this |
7 7 | Total Volume (μl) | 50 |
* Recommended maximum of 1µg of substrate per 10 units of enzyme.
** Restriction Enzymes should be added to the mixture last.
- 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.
Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified Restriction Enzyme
See the NEBuffer Activity/Performance Chart with Restriction Enzymes for the incubation temperatures.
This is the Double Digest Protocol with Standard Restriction Enzymes, using a common reaction and same incubation temperature for both enzymes.
See the NEBuffer Activity/Performance Chart with Restriction Enzymes for the incubation temperatures.
NEBcloner will help guide your reaction buffer selection when setting up double digests.
Make sure to measure DNA concentration in order to ensure the addition of 1µg. DNA used is derived from minipreps