Primer Resuspension and Dilution Calculator

Synthesized primers arrive as lyophilized powder, with a single quantity printed on the tube—for example 32.5 nmol or 200 µg. To prepare a usable stock solution you need to calculate how much water to add yourself. A mistake here propagates to every downstream reaction: every primer concentration will be wrong.

This tool calculates resuspension and dilution in one step, and also tells you how many reactions your tube will support.

The math: write the units out and you won’t go wrong

Errors almost always come from units. Expand the definition:

C(µM) = µmol/L = nmol/mL = (nmol / 1000) per µL

So the volume of water to add for the stock is:

V(µL) = n(nmol) × 1000 / C(µM)

The 100 µM special case is all you need to remember: at 100 µM, V = n × 10. A tube containing 32.5 nmol: add 325 µL of water → 100 µM.

Why everyone makes 100 µM stocks

100 µM is the de facto standard for primer stocks, because it satisfies two requirements at once:

• High enough concentration to keep the storage volume small and minimize freeze–thaw cycles;
• Easy dilution arithmetic: dilute 10× to get a 10 µM working stock, then add 1 µL to a 20 µL reaction for a final concentration of 0.5 µM—the most common working concentration for PCR primers.

The whole workflow runs on powers of ten, so you can verify it in your head. That matters more than saving a few pipette steps.

How many reactions does one tube support?

Using 32.5 nmol as an example:

A standard synthesis scale (minimum order) is far more than enough for most experiments. There is no reason to order a larger synthesis just to “have enough”—the real limitation is shelf life, not total quantity.

nmol or µg?

When both are printed on the tube, use nmol.

Converting µg back to nmol requires the molecular weight, and molecular weight depends on the terminal group. Synthesized primers default to 5′-OH, which is lighter than the 5′-phosphate form by one HPO₃ (79.98 g/mol)—a difference of roughly 1.3% for a 20 nt primer. This tool uses the 5′-OH convention, consistent with the molecular weight values in Basic Sequence Statistics.

If you ordered 5′-phosphorylated primers (for ligation, LCR, etc.), use the phosphate-containing molecular weight for the conversion.

Water or TE?

Both are common; the trade-off is:

• TE (10 mM Tris, 0.1–1 mM EDTA) is more stable. EDTA chelates divalent metal ions, inhibiting metal-dependent nucleases. Preferred for long-term frozen storage.
• Water introduces no buffer components, which suits applications sensitive to the downstream reaction mix. However, water pH tends to drift acidic, and acidic conditions promote depurination.

Note that EDTA from TE carries into the reaction. If the primer volume is large and the reaction is sensitive to Mg²⁺ (e.g., some qPCR assays), use low-EDTA TE (0.1 mM EDTA) or plain water.

Centrifuge before opening

Lyophilized powder scatters to the tube walls and cap during shipping. Briefly centrifuge before opening to pellet the powder at the bottom. Opening the cap first loses some material—and you have no way of knowing how much, so any calculated concentration will be wrong. This is the most common and most avoidable systematic error in primer resuspension.

What this tool does not do

It calculates from the labeled quantity only—it does not measure concentration. The labeled nmol is an OD-based estimate made after synthesis and carries its own uncertainty. To determine the actual concentration after resuspension, measure A260—use the Nucleic Acid Concentration Converter.

It does not convert from OD₂₆₀ units. Some suppliers label tubes in OD rather than nmol. Converting OD to nmol requires the sequence-specific extinction coefficient (calculated by the nearest-neighbor method for each sequence individually), which this tool does not implement.

It does not assess primer quality. Resuspension is only the first step after receiving primers. For dimerization and secondary structure, see Primer Dimer Checker; for Tm, see Nearest-Neighbor Tm Calculator.