Serial dilution cheat sheet

Everything you need to plan an N-step dilution ladder from a stock, in one page.

The identities

Starting from a stock $C_0$ , after N uniform D-fold dilutions:

$C_N = \dfrac{C_0}{D^N}$

To reach a target concentration $C_N$ in exactly N steps, solve for the per-step factor:

$D = \left(\dfrac{C_0}{C_N}\right)^{1/N}$

Each step transfers $V_{\text{step}}/D$ of solution from the previous tube into fresh diluent, so the total volume per tube stays fixed at $V_{\text{step}}$ :

$V_{\text{transfer}} = \dfrac{V_{\text{step}}}{D}$

Stock consumed total = $N \cdot V_{\text{transfer}}$ . Diluent used per step = $V_{\text{step}} - V_{\text{transfer}}$ .

Common ladders

Ladder	D per step	Steps to 10⁶-fold	Transfer (per 100 µL step)	Notes
1:2	2	20	50 µL into 50 µL diluent	Antibody titers, IC₅₀ dose-response
1:3	3	12.6 (round up)	33 µL into 67 µL diluent	Uneven; use for dose response with 3-fold steps
1:5	5	8.6	20 µL into 80 µL diluent	Compromise between resolution and step count
1:10	10	6	10 µL into 90 µL diluent	Default choice — clean powers of 10
1:100	100	3	1 µL into 99 µL diluent	1 µL is at the P2 floor — prefer 1:10

The 1:10 ladder is the workhorse: powers of 10 are easy to reason about, transfers stay comfortably in P20 range, and 6 steps hits the 10⁶-fold dilutions common in microbiology (CFU counts) and PCR (standard curves).

Pipette accuracy floors

Every pipette has a low-end below which the CV climbs. Below these values, spread across replicate transfers is ≥ 5 % and you should split the step further.

Pipette	Range	Accurate down to	Do not use below
P2	0.1 – 2 µL	0.2 µL	0.1 µL
P10 (if available)	0.5 – 10 µL	1 µL	0.5 µL
P20	2 – 20 µL	2 µL	1 µL
P100	10 – 100 µL	10 µL	5 µL
P200	20 – 200 µL	20 µL	10 µL
P1000	100 – 1000 µL	100 µL	50 µL

When to serial dilute

Any time a one-step $C_1V_1 = C_2V_2$ would call for a stock volume below the smallest pipette’s accurate floor. Rough test:

$V_1 = C_2 V_2 / C_1$ ≤ 1 µL → serial-dilute.
Total dilution factor $C_0/C_N \geq$ 100× → serial-dilute in ≥ 2 steps.
Total dilution factor $C_0/C_N \geq$ 10⁵× → serial-dilute in ≥ 3 steps.

Choosing N

Two constraints trade off:

Small N → fewer transfers → less pipetting error, but bigger D per step and possibly a below-floor transfer.
Large N → each step is a comfortable D, but every transfer adds ~1 % CV.

Heuristic. Pick the smallest N such that every step’s transfer volume is ≥ 2× the pipette floor. For most 10⁶ dilutions, that lands on N = 6 with D = 10.

Diluent volume trick

For a step volume of $V_{\text{step}}$ and a dilution factor of D:

$V_{\text{diluent}} = V_{\text{step}} - V_{\text{transfer}} = V_{\text{step}} \cdot \left(1 - \dfrac{1}{D}\right)$

For a 1:10 ladder at 100 µL steps: $V_{\text{diluent}} = 100 \cdot 0.9 = 90$ µL. For a 1:2 ladder at 100 µL steps: $V_{\text{diluent}} = 100 \cdot 0.5 = 50$ µL.

Common mistakes

Not vortexing / not changing tips between steps. Carryover of stock down the ladder inflates every subsequent concentration. Change tips.
Pipetting below the pipette floor. A 0.5 µL transfer with a P20 has ≥ 10 % CV. Split into two steps instead.
Mismatched step volumes. If $V_{\text{step}}$ isn’t constant across the ladder, D per step isn’t either, and the analytical formula $C_N = C_0/D^N$ silently breaks. Every step must use the same volume, or you must track each $V_1$ / $V_2$ pair separately.
Not accounting for compound stickiness. Peptides, hydrophobic small molecules, and DMSO stocks can lose 5–20 % to plastic surfaces per step. Consider low-bind tubes or a carrier protein.

Sources

Rainin RC-500 / RC-1000 pipette specifications — accuracy tables
Gilson Pipetman NEO product spec sheet — CV at low-end volumes
Harris, Quantitative Chemical Analysis, 10th ed. (2020), §2-2 — analytical precision

Last updated: 2026-09-18 .