Molecular Tools

Oligo Resuspension Calculator

Find the buffer volume that hydrates a lyophilised primer, probe, or antisense oligo to your chosen stock concentration. The calculator uses the molar definition C = n/V and outputs volumes in µL or mL.

Resuspend a lyophilised oligo

Enter the nmol value from your IDT, Sigma, Eurofins, or Thermo Fisher QC sheet and your target concentration. Results update as you type.

Common synthesis scales

Synthesis yield

Amount of oligo (nmol)

Read this number from the synthesis report or tube label. IDT prints it under "OD" conversion; Sigma prints µmol or nmol directly.

Target stock concentration

Desired concentration

100 µM is the de facto standard for PCR primers. Use 1 mM for ASOs and some pharmacology stocks.

Resuspension recipe

Add 250.0 µL

of nuclease-free water (or 1× TE buffer, pH 8.0) directly to the lyophilised pellet to produce a 100 µM stock.

1.Spin the tube briefly to pull the pellet to the bottom.

2.Add the calculated diluent volume.

3.Vortex 30 seconds, then incubate at 4 °C for 5 minutes.

4.Spin 30 seconds at 13,000 rpm before opening.

Diluent volume

250.0 µL

water or TE

Stock concentration

100

µM

Total moles

nmol

Aliquot suggestion

× 10 µL tubes

Calculation

Concentration = moles / volume

V = n / C

V = 25 nmol / 100 µM

1 µM = 1 nmol per mL, so V in mL = n (nmol) / C (µM)

V = 250.0 µL

Bench notes

✓Resuspension volume sits in the standard pipettable range.

✓Vortex 30 seconds, spin 30 seconds at 13,000 rpm, then equilibrate at 4 °C for 5 minutes before aliquoting.

Figure 1. Conversion of a lyophilised oligonucleotide pellet (n nanomoles, dry) to a liquid molar stock by addition of a calculated diluent volume V. The relationship C = n / V derives directly from the definition of molar concentration. At 100 µM, every nanomole of oligo occupies 10 µL of solution, so a 25 nmol tube dissolves in 250 µL of nuclease-free water or TE buffer. Pellets contain a fine white powder of sodium-salt oligonucleotide produced by lyophilisation under vacuum at −80 °C.

Why oligos ship dry

Synthesised oligonucleotides arrive lyophilised. Vendors freeze-dry the column eluate under vacuum to remove water, leaving a low-mass white pellet that resists nuclease attack and tolerates shipping at room temperature for 1–2 weeks. A liquid oligo stored at the same conditions would slowly degrade through hydrolytic depurination and trace nuclease activity.

Most vendors quantify the dry product by ultraviolet absorbance at 260 nm and report the result in nanomoles. The QC sheet from IDT shows OD₂₆₀, calculated nmol, and µg mass. Sigma-Aldrich reports the same in either nmol or µmol depending on synthesis scale. Yields drop with longer sequences because each coupling step adds ~99% efficiency, compounding over 30–60 cycles into 70–85% total yield.

A useful fact: the synthesis direction goes 3′ to 5′, opposite to natural DNA synthesis. The 3′ nucleotide attaches first to the solid-phase resin (controlled-pore glass), then phosphoramidite monomers add one at a time toward the 5′ end. This explains why 5′ modifications (FAM, biotin, amine linkers) cost more than 3′ modifications — they survive fewer coupling cycles and therefore tolerate more side reactions.

The 100 µM heuristic

Many labs default to 100 µM stocks because the arithmetic is clean. Add ten times the nmol value as microlitres of diluent, and you reach 100 µM. A 25 nmol synthesis becomes 250 µL of stock. A 100 nmol synthesis becomes 1 mL. The relationship comes from the definition 1 µM = 1 nmol/mL = 1 pmol/µL; multiplying by 100 gives 100 µM = 100 nmol/mL = 1 nmol per 10 µL.

Pick 100 µM unless your downstream protocol forces a different concentration. siRNA experiments often want 50 µM stocks because transfection reagents call for that input directly. Antisense oligonucleotide (ASO) pharmacology uses 1 mM or even 10 mM stocks because the doses given to cells or animals require microlitre transfers from milligram-scale tubes. Aptamer studies favour 200 µM for affinity work because target-binding assays use sub-µM final concentrations and a higher stock minimises serial dilution steps.

Worked examples

Example 1: 25 nmol primer at 100 µM

Spec sheet: 25.0 nmol of a 20-mer PCR primer. Target: 100 µM in TE buffer.

V = n / C = 25 nmol / 100 µM = 25 nmol / (100 nmol/mL) = 0.25 mL = 250 µL

Pipette 250 µL of 1× TE pH 8.0 directly into the supplier tube. Vortex 30 seconds. Aliquot into ten 25 µL single-use tubes for −80 °C archive.

Example 2: 100 nmol probe at 1 mM

Spec sheet: 100 nmol of a fluorogenic TaqMan probe. Target: 1 mM stock for repeated assays.

V = 100 nmol / 1 mM. Since 1 mM equals 1 nmol per µL, V = 100 µL

Resuspend in low-EDTA TE (10 mM Tris, 0.1 mM EDTA) to minimise quenching of the FAM fluorophore. Wrap in foil immediately.

Storage and stability

Stocks held at −80 °C lose less than 5% activity per year. Stocks at −20 °C remain functional for 2 years if freeze-thaw stays under 10 cycles. Working dilutions at 4 °C last 1–3 months but degrade faster at neutral or acidic pH. The dominant degradation pathway is depurination — loss of the purine base from adenosine or guanosine, followed by phosphodiester cleavage at the resulting abasic site. EDTA in TE buffer suppresses this by chelating trace Mg²⁺ and Fe³⁺ ions that catalyse the reaction.

Fluorophore-labelled probes need extra care. FAM, HEX, JOE, and the cyanine dyes (Cy3, Cy5) photobleach under fluorescent room lighting in hours to days. Store in amber tubes, wrap in foil, and minimise time outside the freezer. Quenchers like BHQ-1 and BHQ-2 tolerate light better than the reporter dyes they suppress, so the limiting factor is always the reporter.

Modified oligonucleotides — locked nucleic acid (LNA), 2′-O-methyl RNA, phosphorothioate backbones — share the same handling guidance. Phosphorothioate ASOs are notably stable in serum because the sulfur substitution blocks nuclease cleavage; this is why they reach pharmacokinetic profiles useful for therapeutic dosing.

Limitations and caveats

This calculator assumes the nmol value on the QC sheet is accurate. Spectrophotometric quantification by A₂₆₀ carries 5–10% uncertainty because the sequence-specific extinction coefficient depends on base composition and stacking. For absolute molar accuracy, run the resuspended stock on a NanoDrop and recalculate using the actual sequence-derived ε₂₆₀ (see the Oligo Concentration Calculator).

The formula treats lyophilisation salt content as negligible. In practice, the pellet contains residual sodium ions from synthesis ammonium hydroxide cleavage. For oligos used in high-stringency hybridisation (Northern blot probes, FISH), desalt by ethanol precipitation or Sephadex G-25 before resuspension. Adjust the diluent volume to match your final concentration after desalting losses (~5–10%).

Frequently asked questions

How do I resuspend a lyophilised oligo at 100 µM?

Take the nmol value printed on the tube and add 10× that number in microlitres of diluent. A 25 nmol oligo dissolves in 250 µL to reach 100 µM. The rule works because 1 µM means 1 nmol per mL, so 100 µM is 1 nmol per 10 µL. Spin the pellet down, add the diluent, vortex 30 seconds, rest at 4 °C for 5 minutes, then spin again before pipetting.

Should I use water or TE buffer for oligo resuspension?

TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) stabilises oligos for long-term storage. EDTA chelates divalent metal cofactors that nucleases need to cleave phosphodiester bonds, and the buffered pH 8 prevents the depurination that occurs at acidic pH. Nuclease-free water suits short-term working stocks but leaves the oligo unbuffered, so pH can drift toward acidic values after freeze-thaw cycles. Pick TE for archive stocks above 50 µM, water for working dilutions used within a week.

Why does the IDT spec sheet show nmol instead of mass?

Molar amount is the experimentally relevant quantity for hybridisation. Two oligos with the same mass but different lengths contain different numbers of molecules and bind their targets at different stoichiometries. Synthesis yield in nmol is also directly comparable across orders regardless of sequence length, which is why IDT, Sigma, Eurofins, and Thermo all default to reporting nmol on the QC report. The mass in micrograms appears further down the sheet, calculated from sequence-specific molecular weight.

How long can I keep a resuspended oligo stock?

Concentrated stocks at 100 µM in TE buffer are stable for 2 years at −20 °C and 5+ years at −80 °C if freeze-thaw is limited to fewer than 10 cycles. Working dilutions at 10 µM in water last 6–12 months at −20 °C. Modified oligos with fluorophores (FAM, HEX, Cy3, Cy5) photodegrade faster, so store them in amber tubes or wrap in foil. Probes with quencher modifications (BHQ, TAMRA) are more stable than the fluorophore-only species.

Do I need to filter-sterilise the resuspended oligo?

No. The lyophilisation step kills viable microbes, and standard nuclease-free water arrives DNase- and RNase-treated. Filter-sterilisation introduces handling risk and adsorption loss to the filter membrane, which can drop yield by 5–20% for small volumes. Reserve filtration for cell-culture applications where the oligo is added directly to growing cells, in which case use a low-protein-binding 0.22 µm syringe filter and pre-wet the membrane.

Why is my pellet not fully dissolving?

Three causes: insufficient mixing, GC-rich secondary structure, or insoluble cross-linked degradation products. Vortex for a full 30 seconds, then heat the tube to 65 °C for 5 minutes to disrupt internal base pairing. Spin briefly and re-vortex. If a visible insoluble residue persists, it likely represents oligo that aggregated during synthesis or shipping at high temperature. Contact the supplier — most vendors replace orders that fail to resuspend at the stated yield.

How does this differ from the oligo dilution calculator?

Resuspension converts a dry lyophilised pellet (units: nmol) into a liquid stock at a chosen concentration (units: µM or mM). Dilution converts an already-liquid stock to a lower concentration by adding more diluent. The two calculators chain together in a standard lab workflow: resuspend the new tube to a 100 µM master stock with this tool, then use the dilution calculator to prepare 10 µM working aliquots for PCR.

Reference. Recommendations follow vendor handling guides from IDT and storage stability data from Thermo Fisher Scientific. For regulated diagnostic or GMP workflows, follow facility SOPs.