| Possible cause | Recommendations |
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| Inactive enzyme | - Check the enzyme’s expiration date.
- Verify that the enzyme has been stored at –20°C. Below this temperature, the enzyme may freeze and become inactive.
- Avoid multiple freeze-thaw cycles (no more than three cycles). Use a benchtop cooler to store and transport enzymes.
- Do not store the enzyme in a frost-free freezer or the freezer door shelves.
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| Suboptimal digestion protocol | - Follow the manufacturer’s recommended protocol for the restriction enzyme and type of substrate DNA.
- Verify that all additives or cofactors (e.g., DTT, Mg2+, ATP, S-adenosylmethionine) are present in the reaction. Some restriction enzymes require these for activity (e.g., DTT for Esp3I).
- Use the recommended reaction buffer supplied with the enzyme. For double digestions, follow the manufacturer’s recommendations for buffer compatibility and other reaction conditions. Alternatively, use restriction enzymes that are designed with a single buffer for complete digestion using multiple enzymes.
- Perform the reaction at the optimal temperature specified by the manufacturer. For double digestions with enzymes requiring different temperatures, complete the lower-temperature digestion first, and then perform the second digestion at the higher temperature.
- Ensure that evaporation did not reduce the reaction volume during incubation, leading to increased salt concentrations that may lower enzyme activity. For thermophilic enzymes, use a thermal cycler with a heating lid.
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| Improper dilution of enzymes | - Avoid pipetting small volumes (<0.5 µL). Make a larger working stock to ensure that the enzyme amount added to each reaction is accurate.
- Make the working stock of the enzyme using a dilution buffer recommended by the manufacturer.
- Do not dilute restriction enzymes in water or 10X reaction buffer.
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| Improper reaction assembly | - Add the restriction enzyme last to the reaction. Flick or mix the reaction tube after adding the enzyme to ensure the enzyme does not settle to the bottom due to the glycerol’s density.
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| Excess glycerol in reaction mixture | - Keep the glycerol concentration in the reaction mixture to <5%. The restriction enzyme volume added to the reaction should not exceed 1/10 of the total reaction volume, especially for double digests.
- Ensure that evaporation did not reduce the reaction volume, resulting in an increased glycerol concentration.
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| Suboptimal DNA concentration | - Keep the DNA concentration in the digestion mixture to the optimal range of 20–100 ng/µL.
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| Contaminants in DNA solution | - Remove residual SDS, EDTA, proteins, salts, or nucleases by silica spin-column purification, or by phenol:chloroform extraction and ethanol precipitation. Wash the DNA pellet with 70% ice-cold ethanol to remove EDTA and salts.
- For digestion of unpurified PCR products, set up the digestion reaction with the PCR mixture being no more than 1/3 of the final reaction volume (e.g., 10 µL PCR mixture in 30 µL total digestion reaction).
- If the DNA was purified using silica or resin suspensions, remove all remaining particles by centrifugation for 10 minutes at ≥10,000 x g. Do not carry over resins when transferring the DNA solution to a new tube.
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| Restriction enzyme recognition sequence missing in the substrate DNA | - Re-check or sequence the DNA template.
- If the recognition sequence was introduced by PCR primers, verify that the primer sequence contains the recognition site and an additional 4–8 bases at the 5’ end.
- Check if the restriction enzyme requires more than one recognition site per target for full activity. Addition of a DNA oligonucleotide containing the recognition sequence, or spermidine, may improve the activity of restriction enzymes that require at least two sites for optimal digestion (e.g., AarI, SfiI).
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| Methylation effects | - Check the restriction enzyme’s methylation sensitivity. If the restriction enzyme is inhibited by methylation of its recognition site, look for a neoschizomer or isoschizomer that is not affected by methylation (e.g., MvaI for EcoRII).
- To avoid DNA methylation, propagate plasmids in E. coli hosts that are dam–/dcm–.
- If the restriction enzyme requires methylation at its recognition sequence for activity (e.g., DpnI or SgeI), propagate plasmids in dcm+/dam+E. coli strains. Alternatively, look for a neoschizomer or isoschizomer of the restriction enzyme that digests unmethylated DNA (e.g., MboI for DpnI).
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| Structure of substrate DNA | - For supercoiled plasmid DNA, in which restriction sites may be buried, use enzymes that have been certified to digest intact plasmids. Add more restriction enzyme (e.g., 5–10 units of enzyme per microgram of DNA) if necessary.
- For linear DNA with restriction sites near the ends, check for additional bases required by the enzymes for complete digestion.
- If performing double digestion within the multiple cloning site (MCS) of a vector, check the proximity of the target sequences and required number of bases between the sites for 100% activity. Digestion with the first enzyme may adversely impact the second enzyme’s activity.
- Check if the restriction enzyme requires more than one recognition site per target for full activity. Addition of a DNA oligonucleotide containing the recognition sequence, or spermidine, may improve the activity of restriction enzymes that require at least two sites for optimal digestion (e.g., AarI, SfiI).
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| Impurities in water | - Centrifuge approximately 1 mL of water (10 minutes, 10,000 x g) to pellet and visualize contaminants that may be present.
- Perform a negative-control digestion with water in place of the enzyme to determine the presence of nucleases or bacterial contaminants in water.
- Consider using fresh nuclease-free, molecular biology–grade water from a reliable commercial source.
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