How It Works
This calculator works in 4 steps.
Enter the stock activity
Type U/µL or U/mL from the tube label.
Enter units per reaction and addition volume
Type the units needed and the volume of working enzyme added per reaction.
Enter the working volume
Type the volume of working dilution to prepare.
Read the volumes
The calculator returns the working activity, the enzyme stock volume, and the buffer volume.
The Formula
The formula sets the working activity from units per reaction and applies C₁V₁ = C₂V₂ in U/µL.
A_work = U_rxn / V_addV_stock = A_work × V_work / A_stock%G_final = %G_stock × V_stock / V_work- A_stock / A_work:
- Stock and working activity (U/µL)
- U_rxn:
- Units per reaction
- V_add:
- Volume added per reaction
- %G:
- Glycerol percent
A 20 U/µL restriction enzyme diluted to 1 U/µL in 20 µL needs 1 µL enzyme and 19 µL dilution buffer. Glycerol drops from 50% to 2.5%.
Conversion Table
The conversion table gives enzyme stock volumes for 5 working activities, with 1 µL added per reaction.
| Stock activity | Units per reaction | Working activity | Working volume | Stock volume |
|---|---|---|---|---|
| 20 U/µL | 1 U per 20 µL | 1 U/µL | 20 µL | 1 µL |
| 10 U/µL | 0.5 U per 50 µL | 0.5 U/µL | 20 µL | 1 µL |
| 5 U/µL | 0.05 U per 10 µL | 0.05 U/µL | 100 µL | 1 µL |
| 100 U/µL | 2 U per 25 µL | 2 U/µL | 100 µL | 2 µL |
| 1 U/µL | 0.01 U per 100 µL | 0.01 U/µL | 100 µL | 1 µL |
What Is the Enzyme Dilution Calculator?
The Enzyme Dilution Calculator is a tool that dilutes an enzyme stock labeled in activity units (U/µL or U/mL) to a specified working activity, so a set volume delivers a set number of units per reaction. One unit (U) is the amount of enzyme that converts a defined amount of substrate per minute under the supplier's stated conditions.
Enzyme stocks often sit in 50% glycerol and are too concentrated for direct addition. A working dilution lets the user add 1 µL to deliver, for example, 0.5 U. The calculator recommends the supplier's dilution buffer and reminds the user to keep diluted enzymes on ice and use them the same day.
Common Uses
There are 5 main uses of this calculator.
Restriction digests
Users dilute enzymes for small-scale digests.
Kinetic assays
Biochemists set enzyme activity in the linear rate range.
Polymerase titration
Users test units per reaction in PCR optimization.
Protease digestion
Proteomics labs set trypsin-to-protein ratios.
Activity standards
QC labs prepare enzyme activity standards.