How to plan a restriction enzyme digest
A restriction digest requires the intended DNA mass, a compatible reaction buffer at its working concentration, enough enzyme activity, and water to reach the final volume.
This planner calculates liquid volumes and flags enzyme volumes above 10% of the reaction, but the selected enzyme manufacturer remains the authority for buffer, incubation, and inactivation conditions.
How to use the restriction digest calculator
- Enter DNA: Provide the desired DNA mass and measured DNA stock concentration.
- Enter enzyme: Provide enzyme volume and the units per microliter shown by the supplier.
- Enter buffer and volume: Use the buffer stock factor and intended final reaction volume.
- Review the recipe: Confirm water is non-negative and check enzyme units and the glycerol warning.
Formula and variables
DNA volume follows mass divided by stock concentration. A buffer stock labeled 10× occupies one tenth of the final reaction volume to reach 1×.
V_DNA = m_DNA / C_DNA; V_buffer = V_total / C_stock; V_water = V_total − ΣV_components- m_DNA — DNA mass
- Mass to digest (ng)
- C_DNA — DNA concentration
- DNA stock concentration (ng/µL)
- V_total — Final volume
- Complete digest volume (µL)
One-microgram plasmid digest
Prepare 20 µL using 1,000 ng DNA at 200 ng/µL, 1 µL enzyme at 10 U/µL, and 10× buffer.
- DNA
- 1,000 ng at 200 ng/µL
- Enzyme
- 1 µL at 10 U/µL
- Buffer
- 10×
- Final volume
- 20 µL
- DNA = 1,000 / 200 = 5 µL
- Buffer = 20 / 10 = 2 µL
- Water = 20 − 5 − 2 − 1 = 12 µL
Result: Use 5 µL DNA, 2 µL buffer, 1 µL enzyme, and 12 µL water.
The reaction contains 10 U enzyme, equal to 10 U per µg DNA.
Understanding your results
Check the calculated setup before pipetting
The recipe is a volume plan, not an enzyme-specific protocol.
- Confirm the chosen enzyme is active in the selected buffer.
- Use the supplier’s recommended incubation temperature and duration.
- Account for every additional reagent before calculating water.
Assumptions
- The buffer should reach 1× in the final reaction.
- Enzyme concentration is entered in activity units per microliter.
- DNA concentration and mass use matching ng and ng/µL units.
Limitations
- Does not select an enzyme, predict fragments, or check recognition sites.
- Does not model methylation sensitivity, star activity, additives, or double-digest compatibility.
- Manufacturer instructions can differ by enzyme and supersede generic guidance.
Common mistakes
- Confusing enzyme activity units with enzyme volume.
- Forgetting that a 10× buffer must be diluted to 1×.
- Ignoring additives when calculating the water volume.
- Using an incompatible buffer or incubation temperature.
Practical use cases
Cloning workflows
Plan analytical or preparative plasmid digests before ligation.
Teaching laboratories
Demonstrate mass, concentration, dilution, and enzyme-activity calculations.
Frequently asked questions
How many units of restriction enzyme should I use?
The appropriate activity depends on the enzyme, DNA type, purity, incubation time, and supplier. NEB describes 5–10 U per µg DNA as common guidance for many one-hour digests, but check the specific product protocol.
Why warn when enzyme exceeds 10% of reaction volume?
Many enzyme storage buffers contain glycerol. NEB advises keeping enzyme volume at or below 10% of total reaction volume to reduce the risk of star activity from excess glycerol.
Sources and review
- Optimizing Restriction Endonuclease Reactions — New England Biolabs. Accessed 2026-07-14.
Reviewed 2026-07-14.