What has to be planned before a plate is touched
A plate layout is a volume budget. Every well needs its volume, every replicate multiplies it, every sample multiplies it again, and every dilution level repeats the whole thing. The number that matters is the total at each concentration, and it has to include the volume that will never make it into a well.
Dead volume is the part people underestimate. Liquid left in a reservoir, in a tip, in the bottom of a tube, adds up to a fixed overhead per dilution that does not scale with the number of wells. Preparing exactly the calculated well volume guarantees coming up short.
Replicates, and why they are not optional
A single well gives a number with no way to tell whether it is a measurement or an artefact. Duplicates let you see disagreement; triplicates let you see which of three is the outlier. The cost is linear in plate space and reagent, and the return is the difference between a result and an anecdote.
Two replicates is the usual minimum for a plate reader assay with good precision, such as a protein quantitation. Three is standard where the biology itself is variable, as in cell viability work.
- •Wells per condition = replicates x samples
- •Total wells = wells per condition x number of dilution levels
- •A 96-well plate also has to hold blanks, standards and controls, which come out of the same 96.
Edge effects and plate layout
The outer wells of a microplate evaporate faster than the interior ones and sit at slightly different temperatures, which shows up as a systematic gradient across the plate rather than as random noise. In long incubations the effect is large enough to swamp a modest treatment effect.
The common mitigations are to leave the perimeter unused and fill it with buffer, to randomise the position of conditions so any gradient becomes noise rather than bias, or both. Either one costs plate space, which is why it belongs in the planning stage rather than being discovered afterwards.
How much stock the run will consume
The stock requirement is the sum, across dilution levels, of the volume of stock feeding each one. A tenfold dilution needs a tenth of its total volume as stock; a hundredfold needs a hundredth. The concentrated end of the series dominates the total, which is why extending a series at the dilute end is nearly free and extending it at the concentrated end is not.
Comparing that total against what a vial holds is the last check before starting. The vial calculator tracks what is left; the order quantity estimator turns a programme of runs into a purchase.
How the assay volumes are calculated
Volume per well scaled by replicates and samples, with a fixed dead volume added per dilution, then the stock requirement derived from each dilution factor.
wells per dilution = replicates x samples volume per dilution = (well volume + dead volume) x replicates x samples target concentration = stock concentration / dilution factor stock needed (per) = volume per dilution / dilution factor diluent needed (per) = volume per dilution - stock needed
- Count the wells. Replicates times samples gives the wells at each concentration. Multiplied by the number of dilution levels, that is the plate footprint of the experiment.
- Add the dead volume per well. The dead volume is added to the well volume before scaling, so the overhead grows with the number of wells being served from one reservoir rather than being a single flat addition.
- Divide the stock concentration by each factor. Gives the concentration at each level. The units are whatever the stock was entered in; the arithmetic is a ratio and does not care.
- Work out the stock feeding each level. The volume needed at a level divided by that level's dilution factor. Summing across levels gives the total stock the run consumes.
- Report diluent as the remainder. Total volume at a level minus the stock going into it. Preparing the diluent first and adding stock to it is both faster and easier to keep track of.
What this method cannot tell you
- •The dead volume is a figure you supply, not a measurement of your equipment. It varies with reservoir geometry, tip type and technique.
- •It assumes each dilution is made directly from the stock. For a chained series, the serial dilution calculator models the volumes correctly and this one does not.
- •It does not reserve wells for blanks, standards or controls, which have to come out of the same plate.
- •It does not model adsorptive losses, which matter most at the dilute end of the series and in low-protein buffers.
Assay prep calculator: frequently asked questions
Replicates times samples gives the wells at each concentration, and multiplying by the number of dilution levels gives the total.
Two replicates, four samples and five dilutions is 40 wells, which fits a 96-well plate with room for controls. Three replicates of the same design is 60, which does not leave much.
The liquid that never reaches a well: what stays in the reservoir, in the pipette tip, and in the bottom of the tube.
It is a fixed overhead per dilution rather than a per-well cost, which means preparing exactly the calculated well volume will always leave the last few wells short. Ten to twenty microlitres per well served is a common working allowance.
Two is the usual minimum, three is standard where the biology itself is variable.
- •Duplicates: shows you when two wells disagree
- •Triplicates: lets you identify which of the three is the outlier
- •More than three: rarely worth the plate space unless the effect is small and the noise is high
Sum, across dilution levels, the volume needed at each level divided by that level's dilution factor.
The concentrated end dominates the total. A tenfold dilution needs a tenth of its volume as stock, while a thousandfold dilution needs a thousandth, so adding more dilute levels costs almost nothing.
The outer wells evaporate faster and sit at slightly different temperatures than the interior ones, producing a systematic gradient across the plate rather than random noise.
In long incubations the gradient can be large enough to swamp a modest treatment effect, and because it is systematic, averaging more replicates does not remove it.
Three approaches, often combined:
- •Leave the perimeter wells unused and fill them with buffer or water
- •Randomise the position of conditions so any gradient becomes noise rather than bias
- •Use a plate lid or sealing film and a humidified incubator
Sacrificing the perimeter costs 36 of a 96-well plate's wells, which is why it needs to be decided during planning.
Directly from the stock keeps errors independent, so a mistake at one level does not propagate. A chained series keeps every transfer at a comfortable volume.
Direct dilution is preferable when the factors are modest and the volumes stay measurable. Beyond about a hundredfold, the chained approach wins. This calculator assumes direct dilution; the serial dilution calculator models the chain.
Follow the assay's protocol. Typical figures are 100 microlitres for an ELISA, 25 for a BCA protein assay and 10 for a Bradford, all in a 96-well format.
The volume affects the optical path length in a plate reader, so changing it changes the absorbance reading even at constant concentration. Keep it consistent across the plate.
For any quantitative assay, yes. A standard curve read on a different plate, or on the same plate on a different day, carries the plate-to-plate and day-to-day variation into your result.
Reserve their wells during planning. They come out of the same 96 as everything else.
The volume arithmetic is identical; only the well count and the typical volumes change. Enter the smaller per-well volume you are actually using.
Edge effects are proportionally worse in 384-well plates because the wells are smaller and the surface-to-volume ratio is higher.
If the protocol is written in molar terms and your stock is in mass terms, convert through molecular weight first with the molarity calculator, then enter the molar figure here.
Mixing mass and molar units between the protocol and the plan is a silent error, because the arithmetic still produces plausible numbers.
Yes, whenever the peptide is delivered in anything other than the assay buffer. A DMSO co-solvent, a preservative or an acidic diluent can each produce an effect of their own.
The vehicle control is the highest concentration of vehicle any treated well sees, with no peptide in it.
As close to use as the workflow allows. Dilute peptide solutions lose material to container surfaces over hours, and the loss is proportionally worst at the dilute end.
If a delay is unavoidable, keep the dilutions cold, in low-binding plasticware, and consider a carrier protein where the assay tolerates one.
Reduce a dimension rather than splitting across plates if you can, because plate-to-plate variation adds a confound that no amount of within-plate replication removes.
- •Fewer dilution levels, chosen around the expected active range
- •Fewer samples per run
- •Duplicates instead of triplicates, if the assay's precision supports it
No. It plans volumes, not recovery. A peptide that adsorbs to plasticware will be present at a lower concentration than the plan states, particularly at the dilute end.
Low-binding plates and tips, and a carrier protein where the assay permits, are the usual mitigations.
It is the total the plan consumes. Have more than that on hand, because the figure assumes nothing is lost, no dilution is repeated and no tube is knocked over.
The vial calculator shows how much a partly used vial still holds, which is the number to compare against.
Related Products
Related Research News
Peptide Pull-Down Assays for Antibody Target Validation
Peptide pull-down assays confirm antibody specificity by immobilizing bait peptides to capture target proteins from crude extracts. These methods use biotinylated peptides bound to streptavidin-coupled magnetic beads, allowing rigorous washing to ensure specific binding. Early validation saves time and resources in drug discovery by eliminating nonspecific binders.
Endotoxin-Free Peptides: Key to Accurate T Cell Assays
Trace endotoxin levels in peptides can distort T cell assay results by triggering non-specific immune responses and false positives. Endotoxins, components of gram-negative bacterial LPS, activate TLR4 and influence various immune cells. Continuous testing since 2016 through certified labs has confirmed negative results for all peptides and pools, ensuring reliable reagents for immunology research.
Peptide Array Synthesis: Formats, Spot Sizes, and Custom Quote Requests
Peptide arrays are powerful tools for high-throughput screening, available in different formats depending on assay type. Standard arrays feature small spots (1-2 mm diameter) supporting up to 1,400 peptides per membrane, while large-spot arrays (5-7 mm) accommodate about 110 peptides for solution assays. Maximum array size is approximately 8 cm x 13 cm. This guide explains how to request a custom quote for your specific peptide array needs.