Please useNEB Double Digest Finderto perform a double digest calculation.

Setting up a Double Digestion

• Choose an NEBuffer that results in the most activity for both enzymes. If star activity is a concern, consider using one of our High Fidelity (HF) enzymes.
• If BSA is required for either enzyme, add it to the double digest reaction (BSA does not inhibit any restriction enzyme).
• Set up reaction according to recommended conditions. The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity. For example, in a 50 µl reaction, the total amount of enzyme added should not exceed 5 µl.
• Incubate at recommended temperature. Overnight double digests should be avoided due to the possibility of star activity.
• 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 kill the first enzyme, add the second enzyme and incubate at the recommended temperature.
• Depending on an enzyme's activity rating in a non-optimal NEBuffer, the number of units or incubation time may be adjusted to compensate for the slower rate of cleavage.

Setting up a Double Digestion with a Unique Buffer (designated “U”)

• Our buffer system has been streamlined, leaving three enzymes that have unique buffers: EcoRI (included in this chart and the Restriction Enzyme Activity Chart), SspI (same buffer composition as EcoRI) and DpnII. In most cases, DpnII requires a sequential digest.

Setting up a Sequential Digestion

• Set up a reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer and incubate to completion.
• Adjust the salt concentration of the reaction (using a small volume of a concentrated salt solution) to approximate the reaction conditions of the second restriction endonuclease.
• Add the second enzyme and incubate to complete the second reaction.
• Alternatively, a spin column can be used to isolate the DNA prior to the second reaction.