Restriction Digest of Plasmid DNA
Background Information
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 .
Protocol: Restriction 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:
- DNA
- Restriction Enzyme(s)
- Buffer
- BSA (if recommended by manufacturer)
- dH2O up to total volume
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:1μg DNA
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.
Tips and FAQ
- Restriction enzymes MUST be placed in an ice bucket immediately after removal from the -20°C freezer because heat can cause the enzymes to denature and lose their function.
- The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut. By definition: one unit of enzyme will cut 1ug of DNA in a 50 μL reaction in 1 hour. Using this ratio, you can use the minimal amount of enzyme for your reaction, but keep in mind it is under ideal conditions with very clean DNA, so using a little more enzyme is advisable. Although containing more units of enzyme than is usually required, it is difficult to pipette less than 0.2-0.5 μL and therefore it is common to use something in this range as a minimum when setting up restriction digests..
- If you are having difficulty finding an enzyme that cuts your vector's multiple cloning site (MCS), but does not cut your insert, you could try using two different enzymes that have compatible sticky ends. See NEB's compatible cohesive ends chart.
- If you cannot find compatible sticky ends, you will need to fill in the overhangs and conduct a blunt end ligation. Use T4 DNA Polymerase or Klenow DNA Polymerase I for 3’ overhang removal and 5’ overhang fill-in.
- If you are using blunt ends or a single enzyme to cut the vector, you will need to use a phosphatase to prevent re-circularization of the vector if you are cloning in an insert. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase) are commonly used. Follow the manufacturer’s instructions.
- If your enzyme did not cut, check to make sure that it isn't methylation sensitive. Plasmids grown in Dam or Dcm methylase positive strains will be resistant to cleavage at these restriction sites.
- Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites. This is due to "Star Activity" and can happen for a variety of reasons, including high glycerol concentration. Learn more at NEB's website.
- If you are digesting a large number of plasmids with the same enzyme(s) (for instance, in a diagnostic digest), you can create a "Master Mix" consisting of all of the reaction components except for the DNA. Aliquot your DNA into individual tubes and then add the appropriate amount of Master Mix to each tube. This will save you time and ensure consistency across the reactions.