Many experiments test a compound at several concentrations. A well-planned series covers the right range with sensible spacing, keeps every pipetting step accurate, and keeps everything except the compound constant.
Choosing the spacing
| Spacing | Factor between points | Points per 10-fold | Good for |
|---|---|---|---|
| 2-fold | 2 |
≈ 3.3 |
Detailed curves over a narrow range |
| Half-log | √10 ≈ 3.16 |
2 |
Dose–response curves over several decades |
| 10-fold | 10 |
1 |
Range finding, first experiments |
| Linear | Equal steps | — | Standard curves within a linear range |
Dose–response behaviour is usually logarithmic, so equal ratios (2-fold, half-log) spread points evenly across a sigmoid curve. Linear spacing suits calibration curves where the response is linear in concentration.
Direct or serial?
- Direct dilution makes each tube from the same stock. Errors do not accumulate, but the lowest concentrations need very small stock volumes.
- Serial dilution makes each tube from the previous one. Volumes stay comfortable, but pipetting errors compound down the series.
- Intermediate stocks combine both: dilute the stock once or twice to intermediates, then make each working solution directly from the nearest intermediate.
Keep pipetting volumes in range
For each tube, the stock volume is:
Vstock = Ctarget × Vfinal / Cstock
The worksheet flags any transfer below 2 µL, because volumes that small are hard to deliver accurately with standard pipettes. Fix flagged rows by making an intermediate stock (for example 10-fold) and recalculating those tubes from it, or by increasing the final volume.
Worked example
Stock 10 mM, final volume 1 mL, targets 100, 50, 25, 10, 5 and 1 µM.
Stock volumes: 10, 5, 2.5, 1, 0.5 and 0.1 µL. The last three are below 2 µL. Make a 100 µM intermediate (10 µL of stock + 990 µL of diluent), then prepare 10, 5 and 1 µM from it with 100, 50 and 10 µL in 1 mL.
Keep the vehicle constant
If the stock is in DMSO or another co-solvent, diluting it directly gives every tube a different solvent concentration. Solvents can affect cells and assays on their own, so a concentration effect becomes a solvent effect. Two common fixes:
- Make the dilution series in the co-solvent first, then add the same small volume of each to the assay buffer, so every tube gets the same solvent fraction.
- Add solvent to the lower-concentration tubes so they match the highest one.
Include a vehicle-only control at the same solvent concentration.
Before you start
- Check that the highest target is below the stock concentration and within solubility in the final buffer.
- Prepare 10–20% more volume than you need, to allow for pipetting losses.
- Print the worksheet, tick off each tube as you go, and keep it with your notebook.