What is a double digestion?
A double digest is one where two restriction enzymes are used to digest DNA in a single reaction.
How do you set up double digestion?
If two different incubation temperatures are necessary, choose the optimal reaction buffer and set up reaction accordingly. Add the first enzyme and incubate at the desired temperature. Then, heat inactivate the first enzyme, add the second enzyme and incubate at the recommended temperature.
What is the purpose of double digestion?
Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration.
What is single digestion and double digestion?
Single-digested plasmids are digested with a single restriction enzyme, which results in compatible ends with self-ligation. On the other hand, double-digested plasmids are digested with two different restriction enzymes. Therefore, the two ends are not compatible for self-ligation.
How long do restriction enzymes last?
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 product page for storage information.
How to set up a Double Digest with Neb?
Also, NEB’s online tool NEBcloner ® will help guide your reaction buffer selection when setting up double digests. In most cases, double digests with NEB’s restriction enzymes can be set up in rCutSmart Buffer. Otherwise, choose an NEBuffer that results in the most activity for both enzymes.
What kind of buffer is needed for neb double digestion?
The Performance Chart for Restriction Enzymes indicates if star activity is an issue with sub-optimal buffers. Setting up a Double Digestion with a Unique Buffer (designated “U”) NEB currently supplies three enzymes with unique buffers: EcoRI, SspI and DpnII. In most cases, DpnII requires a sequential digest.
What is performance chart for restriction enzymes in Neb?
The Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers. NEB’s online tools, NEBcloner and Double Digest Finder will help guide your reaction buffer selection when setting up double digests.
How many restriction enzymes are used in double digestion?
Over 215 restriction enzymes are 100% active in rCutSmart™ Buffer, making double digestion simple. If you are using an enzyme that is not supplied with rCutSmart the Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers.