I. Establishing a standard digestion reaction
Most researchers follow a general guideline that 10 units of an endonuclease can effectively cleave 1 μg of DNA, regardless of its source or purity. Typically, in a 50 μl reaction, 1 μl of enzyme is sufficient to digest 1 μg of purified DNA within 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 generally not beyond 16 hours.
Choosing the right enzyme is crucial. The selected enzyme must have at least one recognition site on the target DNA. Enzymes that recognize shorter sequences (e.g., 4 bases) will cut more frequently compared to those recognizing longer sequences (e.g., 6 bases). For instance, a 4-base cutter would cut once every 256 bases on a DNA with 50% GC content, while a 6-base cutter would do so once every 4,096 bases. The resulting fragments may be either sticky ends or blunt ends. Sticky ends can be ligated to compatible fragments, while blunt ends can be joined to other blunt ends. Refer to the "Compatible Cohesive Ends and Recleavable Blunt Ends" catalog for detailed information.
Once the enzyme is removed from the freezer, it should be kept on ice immediately. It’s best to add the enzyme last after all other components are mixed. The amount of enzyme depends on how often it cuts the DNA. For example, supercoiled or trapped DNA may require more than 1 unit per microgram to be fully digested. For more guidance, check "Cutting Plasmid DNA" and "Embedding DNA Cutting" in Tables 244 and 264.
The DNA being digested should not contain contaminants like phenol, chloroform, ethanol, EDTA, detergents, or excessive salts, as these can inhibit enzyme activity. Additionally, DNA methylation can affect digestion efficiency. For more details on methylation, refer to pages 252–253.
II. Buffer and BSA
Each enzyme has an optimal buffer provided by NEB, ensuring nearly 100% activity. Use a 1X buffer concentration. Some enzymes require 100 μg/ml of BSA for maximum performance. In such cases, 100X BSA (10 mg/ml) is available. Adding BSA to enzymes that don’t require it won’t cause harm. For more on buffers, see page 234.
III. Reaction Volume and Glycerol
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 concentration below 5%, ensure the enzyme volume doesn’t exceed 10% of the total reaction (as most enzymes are stored in 50% glycerol).
IV. Mixing
Proper mixing is essential but often overlooked. The reaction mixture should be thoroughly combined. You can use a pipette to mix repeatedly or flick the tube with your finger, then briefly centrifuge. Avoid shaking the tube.
V. Temperature
Most endonucleases work best at 37°C, while thermophilic enzymes require higher temperatures, typically between 50–65°C. Check page 244 for details on thermophilic enzyme activity at 37°C. Always consult the catalog for specific instructions.
VI. Reaction Time
The standard reaction time is 1 hour. If more enzyme is used, the time can be shortened; if less is used, the time can be extended. For more information on enzyme stability during reactions, see page 241.
VII. 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 at 65°C or 85°C for 20 minutes can be used. Not all enzymes are heat-inactivated, so check the Thermal Inactivation Table on page 240. Phenol/chloroform extraction is another option.
VIII. Storage
Most enzymes should be stored at -20°C. A few require -70°C for long-term storage. Always check the data sheet for specific instructions. 10X Buffer and 100X BSA are also stored at -20°C. Never mix BSA with NEBuffer, as this can cause precipitation.
IX. Stability
Enzymes are tested for activity every 1–2 months, and the latest results are listed on each enzyme vial. Based on over 30 years of experience, most enzymes remain stable at -20°C in their recommended buffer. Their activity decreases above this temperature.
X. Control Reactions
If your DNA isn’t cutting properly, run a control experiment. Include a known control DNA with multiple cleavage sites alongside your sample, without the enzyme. If the control DNA is digested, your sample may be contaminated. If the control DNA is cut but your sample isn’t, the issue may be with the enzyme or an inhibitor in your sample. Test again with a mix to confirm.
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