Which strategy should I use?

Equimolar if the reads should be shared evenly. Equal volume is fine when the concentrations are already similar and you want less handling. Weighted when some library needs more reads than the others. Dilute-then-pool when one library is so concentrated that equimolar asks for a fraction of a microlitre.

660 is the NGS convention, so it is the default here. 650 is widely used for dsDNA generally. The two differ by about 1.5%, which rarely changes a decision, but follow your kit insert or your lab's standard if either names a value.

It says a volume is below the minimum.

That library is much more concentrated than the others, so a tiny volume already covers its share. Dilute it first, increase the final volume, or use dilute-then-pool. The tool will not choose for you.

It says the target cannot be reached.

Even taking everything you can, the volumes do not fit inside the final volume at that concentration. The highest reachable concentration for this set is shown next to it — aim at or below that, concentrate the weakest library, or reduce the final volume.

Why does dilute-then-pool have two kinds of warning?

Because it has two steps and two sets of constraints. Taking too little stock at the dilution step and taking too little — or more than exists — at the pooling step are fixed differently: the first by making the diluted volume larger, the second by adjusting the target or the final volume.

Where do my values go?

Nowhere. Everything is computed inside your browser and nothing you type is stored.

NGS library pooling calculator

Volumes to take from each library and buffer to add, for equimolar, equal-volume and weighted pooling.Paste each library's name, length in bp and concentration in ng/µL, give a target pool concentration and a final volume, and get the volume to take from every tube plus the buffer to top up with.

Pooling strategy

Every library contributes the same number of molecules. The default when the reads should be shared evenly.

Libraries

One library per line, as name · length (bp) · concentration (ng/µL). Copying three columns straight out of a spreadsheet is the surest way. Two columns without a name are numbered by row. Up to 384 libraries.

Target pool concentration

Final volume

µL

Molecular weight per bp

g/mol/bp

660 is the NGS convention. 650 is also widely used for dsDNA, and the choice moves the molarity by about 1.5%.

Minimum pipetting volume

µL

Any plan that asks for less than this is flagged. Use the smallest volume you can pipette accurately.

How to use this

Choose a pooling strategy. Equimolar is the default if you are unsure.
Copy three columns — name, length (bp), concentration (ng/µL) — out of a spreadsheet and paste them into the box. For weighted pooling add a fourth column of ratios.
Enter the target pool concentration (nM) and the final volume (µL). Use whatever the sequencer and the library kit ask for.
The molecular weight per bp defaults to 660. Change it if your kit or your lab's standard says 650.
Put the smallest volume you can pipette accurately into the minimum volume field. Anything below it is flagged in the table.
Work down the table taking the stated volume from each library, then add buffer to reach the final volume.

Formulas and cautions

The four strategies differ in one thing only: what share of the pool's molecules each library provides. Once that share is fixed, the arithmetic is the same.

Concentration to molarity

ng/µL is a mass concentration and cannot be added up directly: at the same ng/µL, a library twice as long has half as many molecules.

Molarity (nM): ng/µL × 10⁶ ÷ (length in bp × MW per bp)
e.g. 1 kb · 1 ng/µL · 660: 1 × 10⁶ ÷ (1000 × 660) ≈ 1.515 nM

Volume to take

If library i provides a share wᵢ of the pool's molecules, the volume follows.

Volume (µL): vᵢ = wᵢ × target × final volume ÷ molarityᵢ
Buffer (µL): final volume − Σvᵢ

The share wᵢ in each strategy

Strategy	wᵢ	Result
Equimolar	1/n	every library contributes the same number of molecules
Equal volume	molarityᵢ ÷ Σ molarity	the volume taken is the same from every tube
Weighted	ratioᵢ ÷ Σ ratio	molecules are split in the ratio given
Dilute, then equal volume	1/n (after dilution)	everything is at one concentration, so equimolar and equal volume coincide

Highest reachable concentration

The ceiling is the point where the volumes exactly fill the final volume — where the buffer reaches zero. Nothing above it can be made from this set.

Maximum target (nM): 1 ÷ Σ(wᵢ ÷ molarityᵢ)
Equimolar: the harmonic mean of the molarities
Equal volume: the arithmetic mean of the molarities

Cautions

660 g/mol per bp is the NGS convention. 650 is widely used for dsDNA generally and the two differ by about 1.5% — follow your lab's standard if it has one. This site's ng ↔ pmol converter defaults to 650.
The length is the final library length including adapters. Entering the insert length alone makes the molarity come out too high.
This arithmetic assumes every library is intact double-stranded DNA. Adapter dimers and fragments inflate a fluorometric reading, and the real cluster count then falls short of the calculation. qPCR quantification is more robust to that.
Because the ceiling is a harmonic mean, one very dilute library drags it down on its own. If the target is out of reach, look there first.
This tool reports and does not fix. It will not drop a library or lower the target for you — which library matters is something only you know.
Volumes are shown to two decimal places. The calculation itself is unrounded; rounding happens only where the number is printed.