Pipetting & serial dilution
A 10-fold step means a final volume ten times the transfer: 100 µL into 900 µL of diluent, not into 1000 µL. Adding one volume to ten gives an 11-fold step, which looks like a rounding detail and compounds to nearly two-fold over seven steps. Larger transfers are more accurate because the pipette's error is a roughly fixed number of microlitres.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
10-fold series
A 10-fold step means a final volume ten times the transfer: 100 µL into 900 µL of diluent, not into 1000 µL. Adding one volume to ten gives an 11-fold step, which looks like a rounding detail and compounds to nearly two-fold over seven steps. Larger transfers are more accurate because the pipette's error is a roughly fixed number of microlitres.
How it differs from the standard method
- Prefer 100 µL into 900 µL over 10 µL into 90 µL: the same dilution, roughly a tenth of the relative error, for ten times the reagent.
- A fresh tip for every step: a used tip carries about a microlitre of the previous, ten-fold stronger tube into the next one.
- Vortex each tube before taking the next aliquot. An unmixed aliquot sits as a layer on top of the diluent for minutes, and the next transfer comes from that layer.
- Dip the tip only 2–3 mm: liquid clinging to the outside of a deeply immersed tip is carried forward on top of the measured volume.
- For very large dilution factors, two accurate 100-fold steps beat four sloppy 10-fold ones; each step you remove is one less chance to compound.
1What a pipette actually measures
An air-displacement pipette moves a piston through a sealed shaft. The piston displaces a volume of air, and that column of air pulls liquid into a disposable tip.
Error is absolute
A pipette's specification is tightest at its nominal volume and worst at the bottom of its range. The same 20 µL is excellent on a P20 and poor on a P200.
Reverse mode
Aspirate past the set volume, dispense only to the first stop and throw the rest away with the tip. The volume is then the piston stroke alone, and the film that stays behind stops mattering.
Carry-over
A used tip holds about a microlitre inside it. Carried into a tube ten times weaker, that microlitre is a large addition of the stronger solution.
2How error travels down a dilution series
A serial dilution multiplies: each tube is made from the one before it, so each tube inherits every error that came before. The two kinds of error behave completely differently.
- Random error
- Scatter between repeats of the same operation. Visible in replicates; grows as √n down a series; reduced by averaging.
- Systematic error
- A bias in the same direction every time. Invisible in replicates; compounds down a series; only an external reference finds it.
3Laying out a plate
A 96-well plate adds geometry to the problem. The 36 wells of the outer ring evaporate faster and sit at a slightly different temperature from the 60 inside, so in any assay that runs for half an hour they drift away from the interior: the edge effect.
Key terms
Practise these as flashcards →The difference between the mean volume delivered and the volume set. Compounds down a dilution series and is invisible in replicates.
The scatter of repeated dispenses, usually quoted as a CV. Adds in quadrature, so it grows as the square root of the number of steps.
Weighing repeated dispenses of water on a balance and converting mass to volume with the liquid's density, to measure a pipette's accuracy and precision.
Aspirating more than the set volume and dispensing only to the first stop, so the volume delivered is the piston stroke and the retained film is discarded.
Aspirating and dispensing the liquid two or three times with a new tip, to coat its wall and saturate the air cushion with vapour before the first real transfer.
Liquid brought from one vessel into the next on or in the tip. In a dilution series it always moves material from the stronger tube to the weaker one.
The systematic difference between the outer ring of a microplate and its interior, caused mainly by faster evaporation and a temperature gradient.
Standard deviation divided by the mean, as a percentage. The usual way to quote replicate agreement in a plate assay.
When to use it
- Building standards for any quantitative assay: a curve is only as good as the series that made it, and the bottom of the curve is where dilution errors show up first.
- Diluting a sample into the working range of an assay, where the dilution factor multiplies both the result and its error.
- Counting by plating or by limiting dilution, where 10-fold series over six or more decades are the measurement itself.
- Any time a protocol asks for a transfer under 10 µL, which is where the choice of pipette and technique stops being a detail.
Compared with alternatives
Where the stocks and diluents come from; use it when the problem is the solution's composition rather than the volume transferred.
The assay a standard series usually feeds. Go there for the immunochemistry; come back here when the bottom of the curve misbehaves.
The same 10-fold series, but the readout is colonies, so Poisson counting noise rather than pipetting sets the precision.
Reading the plate you have just filled; prefer it when the question is optics, gain and well-to-well crosstalk rather than volume.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A 10-fold step means a final volume ten times the transfer: 100 µL into 900 µL of diluent, not into 1000 µL. Adding one volume to ten gives an 11-fold step, which looks like a rounding detail and compounds to nearly two-fold over seven steps. Larger transfers are more accurate because the pipette's error is a roughly fixed number of microlitres.
- Prefer 100 µL into 900 µL over 10 µL into 90 µL: the same dilution, roughly a tenth of the relative error, for ten times the reagent.
- A fresh tip for every step: a used tip carries about a microlitre of the previous, ten-fold stronger tube into the next one.
- Vortex each tube before taking the next aliquot. An unmixed aliquot sits as a layer on top of the diluent for minutes, and the next transfer comes from that layer.
- Dip the tip only 2–3 mm: liquid clinging to the outside of a deeply immersed tip is carried forward on top of the measured volume.
- For very large dilution factors, two accurate 100-fold steps beat four sloppy 10-fold ones; each step you remove is one less chance to compound.