I. Establishing a standard digestion reaction
Most researchers follow a general guideline where 10 units of an endonuclease can digest 1 μg of DNA, regardless of its origin or purity. In a typical 50 μl reaction, 1 μl of enzyme is sufficient to cleave 1 μg of purified DNA in 1 hour under optimal conditions, such as 1X NEBuffer and the appropriate temperature. If more enzyme is used, the reaction time can be reduced; conversely, if less enzyme is added, the time can be extended, though not beyond 16 hours for many enzymes.
Choosing the right enzyme is crucial—each enzyme must have at least one recognition site on the target DNA. Enzymes that recognize fewer bases (e.g., 4 bp) cut more frequently than those recognizing more (e.g., 6 bp). For example, in a DNA with 50% GC content, a 4-base cutter will cut once every 256 bases, while a 6-base cutter will do so once every 4096 bases. The resulting fragments may be sticky ends (3’ or 5’ overhangs) or blunt ends. Sticky ends are compatible with other cohesive ends, while blunt ends can ligate together. Refer to the Compatible Cohesive Ends and Recleavable Blunt Ends catalog for details.
Once the enzyme is removed from the freezer, it should be kept on ice immediately. It’s best to add the enzyme last to the reaction mix, after all other components have been pre-mixed. The amount of enzyme depends on how often it cuts the DNA. Supercoiled or trapped DNA usually requires more than 1 U/μg for complete digestion. For specific guidance, refer to “Cutting Plasmid DNA†and “Embedding DNA Cutting†in Tables 244 and 264.
The DNA being digested should be free from contaminants like phenol, chloroform, ethanol, EDTA, or high salt concentrations, which can inhibit enzyme activity. Methylation status is also important—check pages 252–253 for methylation-related information.
II. Buffer selection
Each enzyme comes with a recommended NEB buffer that ensures near-100% activity. Use 1X buffer concentration. Some enzymes require 100 μg/ml BSA for optimal performance. In such cases, 100X BSA (10 mg/ml) is provided. Adding BSA to enzymes that don’t need it won’t harm them. For more on buffers, see page 234.
III. Reaction volume and glycerol concentration
An enzyme unit is defined as the amount needed to digest 1 μg of DNA in 50 μl within 1 hour. This helps determine the enzyme-to-DNA ratio. Smaller volumes are more prone to pipetting errors. To keep glycerol below 5%, ensure the enzyme volume does not exceed 10% of the total, as most enzymes are stored in 50% glycerol.
IV. Mixing the reaction
Mixing is a critical but often overlooked step. Ensure thorough mixing by pipetting up and down several times, flicking the tube, and briefly centrifuging. Avoid shaking or vortexing, as this can damage the enzyme.
V. Temperature and incubation time
Most enzymes work best at 37°C, while thermophilic enzymes require higher temperatures (typically 50–65°C). Check page 244 for details on thermophilic enzyme activity at 37°C. Always consult the enzyme catalog for specific requirements.
VI. Stopping the reaction
If no further digestion is planned, stop the reaction using a termination solution: 50% glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue (10 μl per 50 μl reaction). Alternatively, heat inactivation (65°C or 85°C for 20 minutes) can be used, though not all enzymes are affected this way. Phenol/chloroform extraction is another option. See page 240 for thermal inactivation details.
VII. Storage
Most enzymes should be stored at -20°C. A few require long-term storage at -70°C. Check the data sheet for specifics. 10X buffer and 100X BSA are also stored at -20°C. Never mix BSA with NEBuffer, as this can cause precipitation.
VIII. Stability and quality control
Enzymes are tested for activity every 1–2 months, and test results are listed on each tube. Over 30 years of experience shows that most enzymes remain stable at -20°C in their recommended buffer. Stability decreases above -20°C.
IX. Control experiments
If your DNA isn’t cutting as expected, run a control experiment. Include the substrate DNA without enzyme, and compare it with control DNA containing known sites. If the control DNA is cleaved but the sample is not, the issue may be an inhibitor (like salt, EDTA, or phenol) in your sample. Try re-running the reaction with both DNAs to confirm.
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