When carrying out molecular cloning, sooner or later, you will need to do restriction digests - to excise a fragment of interest from your source DNA, linearize your plasmid vector or to check positive clones. During this technique the DNA is cleaved at specific recognition sites by restriction enzymes.
However, the restriction digest can fail for a variety of reasons, the critical factors are; the composition of the buffer, incubation temperature, DNA methylation, star activity, multiple digestion steps as well as the DNA substrate itself.
If this is the case for you, don’t worry. This ZAGENO troubleshooting guide is here to help you! In the following section, you find some of the most common problems and their solutions.
Incomplete digestion or no digestion
- Salt Inhibition: Some enzymes are less active in the presence of high salt concentrations. Evaporation during the incubation can increase the salt concentration and affect the enzyme activity. Clean up your DNA prior to digestion.
- Impure DNA: DNA preparations may contain other contaminants like phenol, chloroform and ethanol, that partially or completely inhibit the enzyme activity. Clean up your DNA prior to digestion.
- Using the wrong buffer: Use a recommended buffer supplied with the restriction enzyme. For double digests, follow the manufacturer’s recommendations for optimal buffer compatibility.
- Too few units of enzyme: Use at least 3-5 units of enzyme per µg of DNA
- Incubation time too short: Increase the incubation time
- Inactive enzyme: Check the expiration date and make sure the enzyme was not stored under -20 °C. Below -20 °C the enzyme will freeze – multiple freeze-thaw cycles can also influence the activity of your enzyme.
- Missing additives or cofactors: For optimal enzyme activity, make sure that you add the appropriate cofactors or additives (DTT, Mg2+, ATP).
- Temperature is not optimal: Perform the incubation step at the recommended temperature for the enzyme. When doing double digestions, complete the lower-temperature digestion step first and then perform the one at the higher temperature.
- Improper enzyme dilution: Dilute the enzyme with Dilution Buffer for Restriction enzymes. Never use water or 10X reaction buffer.
- Improper reaction assembly: The enzyme should always be the last component added to the reaction mixture. If the enzyme is added directly to the 10X reaction buffer, it might be inactivated.
- Excess glycerol in the reaction mixture: The glycerol concentration should be less than 5 %.
- DNA concentration not optimal: Ensure that the DNA concentration is in the range of 20-100 ng/µl.
- Recognition sequence of restriction enzyme not in substrate DNA: Re-check the sequence of your template DNA.
- Methylation effects: Check the enzyme's’ sensitivity to methylation. If it is affected by methylation, look for Isoschizomers, which are specific to the same recognition sequence. To avoid DNA methylation, propagate plasmids in E. coli that are dam-/dcm-.
- Incorrect incubation temperature: Most restriction enzymes optimally cut DNA at 37 °C but there are many exceptions. Incubate your digest reaction at the recommended temperature.
- Impurities in water: Perform a negative control containing water, to determine the presence of nucleases or bacterial contaminations. Always use fresh, molecular biology-grade water.
Unexpected cleavage pattern
- Partial restriction enzyme digests: Contamination with inhibitors, or high salt concentrations can affect the digestion reaction. Clean up the DNA before restriction digest.
- Star activity: In some cases restriction enzymes cleave sequences which are similar, but not identical to their defined recognition sequence. Reduce the units of the restriction enzyme and use the recommended reaction buffer.
- Contamination with another enzyme: Your buffer or enzyme might be contaminated with another enzyme. Use a fresh tube of buffer and/or enzyme.
- Contamination with other substrate DNA: The sample DNA contains more than one sequence of DNA. Prepare a new sample of DNA.
- Unexpected recognition sequences in substrate DNA: Re-check your cloning strategy and the sequence of your template DNA.
Diffuse DNA bands
- Poor DNA quality: Check the undigested DNA by electrophoresis. If a smear is observed on the gel, re-purify the DNA by spin column or phenol/chloroform extraction and ethanol precipitation.
- Contaminated reagents: Reaction components may have been contaminated with nucleases from improper handling, which causes DNA degradation. Prepare new reagents, use new enzymes and buffers, or re-purify DNA if necessary.
- Place the enzyme immediately on ice after removal from -20°C. Heat can cause denaturation of the enzyme and a loss of its function.
- The amount of restriction enzyme depends on the amount of DNA you want to cut. In general: one unit of enzyme cuts 1 µg DNA in a 50 µl reaction in 1 hour.
- Make sure the enzyme is the last component that is added to the reaction mixture.
- Do not vortex the restriction enzyme, just mix by flicking or pipetting up and down.
- Perform a digestion reaction with a standard control DNA, e.g. Lambda DNA
- Always check the reaction conditions - temperature, incubation time, buffer compatibility - which are recommended by the manufacturer.
- Check the DNA on a gel. Also, load the undigested DNA. Then you can be sure that something went wrong in the digestion process and nothing is wrong with your DNA.
We hope that by following these tips, your next restriction enzyme digestion will be successful. For problems not added here or some tips - let us and the community know in the comments!