For many quantification assays, a set of standards must be run alongside test samples in order to calibrate an experiment properly. These standards are used to create what is known as a calibration curve, or standard curve. From this curve, the quantity of target in a sample can be calculated.
Standards for the calibration curve are typically chosen such that they span the linearity of a given assay. This represents the range of quantities over which response values are directly proportional to changes in the target of interest. For many quantification assays, the linearity range is logarithmic in nature. Thus, when creating standards for a given assay, it is often necessary to prepare the standards using a serial dilution.
A serial dilution is a sequence of dilutions created using the same dilution factor. For instance, creating a two-fold dilution with a starting concentration of 10 µM yields the following concentrations: 10 µM, 5 µM, 2.5 µM, 1.25 µM, etc.
The tool below can be used to create a protocol for preparing a serial dilution from a stock solution.
| Calculate serial dilution using | Initial concentration and dilution factor |
| Stock solution name | |
| Diluent name | |
| Concentration of stock solution | µM |
| Serial dilution initial concentration | µM |
| Dilution factor | |
| Final volume of each dilution | µL |
| Number of dilutions | |
| Number of replicates (optional) |
| Dilution | NADH standard Volume (µL) | Source | PBS buffer Volume (µL) | Concentration (µM) |
|---|---|---|---|---|
| 1 | 50 | from 30 µM stock | 100 | 10 |
| 2 | 50 | from dilution 1 | 100 | 3.3333 |
| 3 | 50 | from dilution 2 | 100 | 1.1111 |
| 4 | 50 | from dilution 3 | 100 | 0.37037 |
| 5 | 50 | from dilution 4 | 100 | 0.12346 |
| 6 | 50 | from dilution 5 | 100 | 0.041152 |
| 7 | 50 | from dilution 6 | 100 | 0.013717 |
| Control | 0 | from PBS buffer | 100 | 0 |
- Pipette 50 µL of 30 µM stock into 100 µL of PBS buffer . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 1 (from step 1) into 100 µL of PBS buffer to create dilution 2 . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 2 (from step 2) into 100 µL of PBS buffer to create dilution 3 . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 3 (from step 3) into 100 µL of PBS buffer to create dilution 4 . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 4 (from step 4) into 100 µL of PBS buffer to create dilution 5 . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 5 (from step 5) into 100 µL of PBS buffer to create dilution 6 . Mix well before continuing. Avoid generating bubbles.
- Pipette 50 µL of dilution 6 (from step 6) into 100 µL of PBS buffer to create dilution 7 . Mix well before continuing. Avoid generating bubbles. Discard 50 µL from dilution 7 to obtain the correct volume for the final dilution.
- Create a blank control using 100 µL of PBS buffer. This should be enough for 2 replicates.
- Using the table in the protocol as a guide, pipette 25 µL of each standard into its corresponding well in the experimental microplate. Standards prepared with this protocol should be enough for 2 replicates. Use of a multi-channel pipette is highly recommended.
MLA | "Quest Calculate™ Serial Dilution Calculator and Planner." AAT Bioquest, Inc., 27 Sep. 2025, https://www.aatbio.com/tools/serial-dilution. | |
APA | AAT Bioquest, Inc. (2025, September 27). Quest Calculate™ Serial Dilution Calculator and Planner. AAT Bioquest. https://www.aatbio.com/tools/serial-dilution. | |
| BibTeX | EndNote | RefMan |
- Dilution Factor
Calculate dilution factor given initial and final volumes
- M1V1 = M2V2
Solve for concentration or volume with a dilution
- Serial Dilution
Generates a serial dilution protocol based on dilution factor or concentration range