NGS library concentration (ng/µL → nM) | dilution & pooling

How to use (3 steps)

Choose an example or enter library concentration (ng/µL or nM) and fragment length (bp).
Select a target nM and final volume to see a dilution recipe (stock + water).
Choose a pooling method and review per-library volumes and pooled nM (guide).

Conversion to nM depends on length, MW factors, and quantification method. Treat results as a guide and follow your protocol for final decisions.

Inputs (library table)

Example (preset)

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Paste (CSV/TSV)

Paste directly from Excel/Sheets (tab or comma-separated). Example columns: name, ng_per_uL, length_bp, nM (column names can vary).

Name	Input mode	ng/µL	Length (bp/nt)	nM (input)	Concentration (nM)	Actions

Table scrolls horizontally (swipe or drag).

Molecule type (dsDNA, etc.)

Molecule type MW factor (g/mol/bp or nt)

dsDNA (660 g/mol/bp) is a common guide for NGS. Values can vary by method and definition.

Normalization (dilute to match nM)

Target nM preset Target concentration (nM)

Final volume preset Final volume (µL)

Pooling

Method

Volume per library (µL)

Results (conversion, dilution, pooling)

Name	Stock (nM)	Dilution: stock (µL)	Dilution: water (µL)	Pool volume (µL)

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How it's calculated

Conversion (guide): nM = (ng/µL) × 1e6 / (length × MW_per_bp)
Dilution: C1V1 = C2V2 (V_stock = (C_target/C_stock) × V_final)
Direct equal-molar pooling: allocate volumes so C_i × V_i is equal (given total volume)

Conversion depends on length, MW factors, and quantification method. Use these results as a guide.

FAQ

What is ng/µL → nM conversion used for?

Use it to normalize libraries by molecule counts for equal-molar pooling (different from equal mass).

Where do I get fragment length (bp)?

Use the average size from a Bioanalyzer/TapeStation report. Keep adapter inclusion consistent with the report definition.

What is the difference between equal-mass and equal-molar pooling?

Equal mass matches ng values, while equal molar matches molecule counts (nM). If lengths differ, equal mass will not equalize molecule counts.

What is normalization?

Normalize by diluting all libraries to the same nM, then pool equal volumes. This tool provides that recommended flow.

The dilution volume is too small (e.g., 0.2 µL).

Very small volumes are hard to pipette. Consider intermediate dilution or adjusting final volume.

Can I use nM (or pM) values from qPCR?

Yes. Use the direct nM input mode. Note that quantification definitions can differ by method.

Will the pooled concentration be exact?

It is a guide. Quantification and pipetting errors and fragment length variation can shift the final value.

Comments

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