How It Works
This calculator works in 4 steps.
Enter the addition volume
Type the volume of the reagent or compound added.
Enter the final well volume
Type the total volume after all additions.
Enter the final concentration
Type the in-well concentration the protocol calls for.
Read the intermediate concentration
The calculator returns the in-well DF and the strength to prepare before adding.
The Formula
The formula multiplies the final well strength by the dilution in the well.
DF = V_final / V_addC_int = C_final × DFC_final = C_int × V_add / V_final- V_add:
- Volume of the addition
- V_final:
- Total well volume after all additions
- C_final:
- Target in-well concentration
- C_int:
- Concentration of the addition
A protocol adds 10 µL compound to cells in 90 µL medium for 100 µL total. For 1 µM in the well, prepare the compound at 10 µM.
Conversion Table
The conversion table gives the intermediate concentration for 6 addition and well volume combinations.
| Addition | Final well volume | In-well DF | Final target | Prepare addition at |
|---|---|---|---|---|
| 10 µL | 100 µL total | 10 | 1 µM | 10 µM |
| 5 µL | 50 µL total | 10 | 10 µM | 100 µM |
| 2 µL | 200 µL total | 100 | 0.5 µM | 50 µM |
| 25 µL | 100 µL total | 4 | 100 µM | 400 µM |
| 1 µL | 100 µL total | 100 | 5 µM | 500 µM |
| 50 µL | 200 µL total | 4 | 2 µM | 8 µM |
What Is the Assay Addition Dilution Calculator?
The Assay Addition Dilution Calculator is a tool for reagents that get diluted in the well. It tells you how strong to make a reagent before you add it. A 10 µL addition into a 100 µL final well volume is diluted 10-fold.
Protocols give the final strength in the well. But you make each reagent up at a higher, in-between strength. Forgetting the in-well dilution gives final levels 2-fold to 100-fold below plan. It starts from the final well volume, with every part added after the reagent. Then it gives the strength to make up first.
Common Uses
There are 5 main uses of this calculator.
Compound intermediate plates
Screening labs prepare 10× compound plates.
ELISA reagent additions
Users prepare detection reagents at the correct in-well strength.
Enzyme assays
Biochemists set substrate and enzyme additions.
Reporter assays
Cell biologists add stimuli at 5× or 10× strength.
Multi-step additions
Users account for later additions that dilute earlier ones.