Serial Dilution Calculator for 96-Well Plate Transfers
Plan a ten-point threefold concentration series for triplicate wells using the independently checked serial-dilution reference. The live calculation accounts for the material passed to the next tube as well as the volume needed in the wells. It also shows why serial dilution in plain diluent does not maintain a constant DMSO fraction.
The original fixture specifies a 100 µM top concentration, ten points, three 50 µL wells per concentration, and a 10 mM stock in neat DMSO. These are worked preparation inputs rather than an experimentally established protocol for a particular compound.
Connect concentration spacing to physical volumes
The fold factor determines the logarithmic spacing between concentrations, while the replicate count and well volume determine the liquid demand. These are separate choices. Increasing replicates changes preparation volumes without changing the nominal concentration series. Changing the fold factor alters both the concentration spacing and the transfer relationship, so review the entire plan after either edit.
The top tube is prepared from the declared stock. Subsequent tubes receive a fraction of the previous tube and the balance as diluent. The final tube has no further downstream requirement, so the plan exposes its remaining surplus rather than pretending every prepared microlitre is consumed. This is useful when comparing a theoretical minimum with a practical preparation allowance.
Retain the solvent and preparation context
A concentration label alone does not state the solvent composition experienced by the cells or assay reagents. The reference begins with a neat-DMSO stock, so its top concentration has the largest transferred solvent fraction. Lower concentrations carry progressively less stock solvent. Vehicle controls should reflect the experimental design, and an unexplained solvent gradient can confound a compound-response interpretation.
When transferring the plan into a plate map, preserve compound identity, concentration units, well volume and replicate positions. The map records what was intended to be dispensed; the raw plate readings remain separate observations. If precipitation, adsorption or instability is suspected, the nominal dilution series may not describe the actual available concentration. Such effects require experimental investigation rather than a numerical correction guessed from the response curve.
Equations and variables
Concentration series
ci = c0/fⁱ
- ci
- Concentration at zero-based step i (mol/L)
- c0
- Top concentration (mol/L)
- f
- Dilution factor, greater than one
Transfer and retained volume
Vtube = Vwells × f/(f − 1); Vtransfer = Vtube/f
- Vwells
- Volume retained for the specified replicate wells (µL)
- Vtube
- Working tube volume before onward transfer (µL)
- Vtransfer
- Volume transferred to the next dilution (µL)
Scope and limitations
- The solvent fraction falls along this series when the diluent contains no matching solvent. The tool flags and displays fractions; it does not guarantee constant-vehicle backfilling.
- Calculated transfers assume complete mixing, volume additivity and no adsorption, precipitation or evaporation.
- A mathematically valid volume may be outside a pipette’s useful range. Review minimum-transfer flags and any need for an intermediate stock.
Sources and provenance
- Calculator inputs and independent arithmetic verificationOriginal synthetic inputs; see individual fixture notices
- Independent serial-dilution fixture, method version 2Original generated inputs and exact arithmetic reference
Technical questions
Why do I need more liquid than the wells consume?
Each intermediate tube supplies both its own replicate wells and the next dilution. The recurrence reserves an onward-transfer volume before dispensing. Omitting that amount leaves later tubes short even if the nominal concentration ratio is correct.
Is DMSO constant across the ten concentrations?
No. With plain diluent, the solvent from the original stock is diluted together with the compound. If constant vehicle is required, it needs an explicit matched-solvent preparation design and appropriate controls; the current reference does not silently add that backfill.
Can I use the transfer volume as a pipetting instruction without checking?
Check the pipette range, stock availability, mixing procedure and compound behaviour first. The calculator performs mass-balance arithmetic, not a measurement of dispensing accuracy or solubility. Preserve any intermediate dilution and its concentration basis with the plan.
Connected workflows
- 96-well dose-response plate mapping
Carry intended concentrations into a mapped assay plate.
- 4PL IC50 curve fitting
Fit the observations obtained from a concentration series.
- DMSO stock reconstitution volume
Prepare the stock whose concentration the dilution plan assumes.
- Two-Step Intermediate Dilution Planner for Small Volumes
Break a large single-step dilution into two practical transfers above the pipette minimum.