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Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of many commonly used restriction enzymes, visit NEB.
Restriction enzyme digestion is a commonly used technique for molecular cloning, such as in cloning by either PCR or restriction enzyme digest. It is also used to quickly check the identity of a plasmid by diagnostic digest .
1. Select restriction enzymes to digest your plasmid.
Note: To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence analysis program such as Addgene's Sequence Analyzer.
2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
Note: If you are conducting a double digest (digesting with two enzymes at the same time), you will need to determine the best buffer that works for both of your enzymes. Most companies will have a compatibility chart, such as the double digest chart from NEB. If you cannot find a buffer that is appropriate for both of your enzymes, you will need to digest with one enzyme first in the buffer for enzyme 1, repurify the cut plasmid, and then conduct the second digest in the buffer for enzyme 2.
3. In a 1.5mL tube combine the following:
Note: The amount of DNA that you cut depends on your application. Diagnostic digests typically involve ~500ng of DNA, while molecular cloning often requires 1-3μg of DNA. The total reaction volume usually varies from 10-50μL depending on application and is largely determined by the volume of DNA to be cut.
Note: See Tip and FAQ section below for note on determination of restriction enzyme volume to use.
Note: A typical restriction digestion reaction could look like this:
1 μL of each Restriction Enzyme
3μL 10x Buffer
3 μL 10x BSA (if recommended)
x μL dH2O (to bring total volume to 30 μL)
4. Mix gently by pipetting.
5. Incubate tube at appropriate temperature (usually 37°C) for 1 hour. Always follow the manufacturer’s instructions.
Note: Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2hr is often sufficient. For digests with >1μg of DNA used for cloning, it is recommended to digest for at least 4hr.
Note: If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction. This may involve incubating the reaction at 70°C for 15 mins, or purifying the DNA via a purification kit, such as a QIAGEN DNA cleanup kit. See the enzyme manufacturer's instructions for more details.
6. To visualize the results of your digest, conduct gel electrophoresis.