Primer Resuspension & Dilution Calculator
Calculate exact buffer volumes to reconstitute lyophilized primers to 100 µM stock solutions, prepare 10 µM PCR working dilutions, and check primer MW and Tm.
Lyophilized Oligo Details
Check the manufacturer tube label (e.g. 25.4 nmol)
Solvent Volume to Add
How to Use the Primer Resuspension Calculator
- Find the actual amount delivered (nmol) on the tube or specification sheet, not the synthesis scale ordered.
- Choose the stock concentration. 100 µM is a common starting point, not a requirement for every assay.
- Read the solvent volume, select a compatible buffer or nuclease-free water, and follow the supplier’s resuspension instructions.
- Use Working Dilution to prepare a lower-concentration solution. Calculate each primer separately and check the final reaction concentration.
Scientific Principles & Formula
Resuspension volume V (µL) depends on the delivered amount n (nmol) and target concentration C (µM):
Since 1 µM = 1 pmol/µL = 0.001 nmol/µL, a 100 µM concentration equals 100 pmol/µL (0.1 nmol/µL), giving Volume (µL) = nmol × 10.
Worked examples: from dry primer to PCR
Follow one primer through resuspension, working dilution, and reaction setup. These are calculation examples; use the concentrations specified by your assay.
1. Make a 100 µM stock from 32.4 nmol
The tube contains 32.4 nmol of dried primer. Divide by the target concentration and convert nmol to pmol.
V = 32.4 × 1000 / 100 = 324 µL
Add 324 µL of a compatible solvent to the dried primer to make a 100 µM stock.
2. Prepare 100 µL of a 10 µM working solution
Use the 100 µM stock from example 1. The desired 100 µL is the total volume after mixing, not the amount of water to add.
V₁ = C₂V₂ / C₁ = 10 × 100 / 100 = 10 µL
Mix 10 µL of stock with 90 µL of diluent. This is a tenfold dilution: one part stock plus nine parts diluent.
3. Calculate primer volume for a 20 µL reaction
For a final concentration of 0.5 µM of one primer, use the 10 µM working solution from example 2.
V₁ = 0.5 × 20 / 10 = 1 µL
Add 1 µL of that primer. If forward and reverse primers both have this target, add 1 µL of each and include both volumes in the 20 µL reaction total.
Primer resuspension volume reference
| Delivered amount (nmol) | 20 µM | 50 µM | 100 µM |
|---|---|---|---|
| 5 | 250 µL | 100 µL | 50 µL |
| 10 | 500 µL | 200 µL | 100 µL |
| 25 | 1250 µL | 500 µL | 250 µL |
| 50 | 2500 µL | 1000 µL | 500 µL |
| 100 | 5000 µL | 2000 µL | 1000 µL |
Units and common mistakes
- Amount and concentration differ: nmol describes how much primer you have; µM describes amount per volume. 1 µM = 1 pmol/µL, so 100 µM = 100 pmol/µL.
- Stock concentration, working concentration, and final reaction concentration are different. A 10 µM working tube does not make a 10 µM PCR reaction when only a small aliquot is added.
- For dilution, diluent volume = final volume − stock volume. Do not add the full final volume of water on top of the stock.
- If a transfer is below your pipette’s reliable range, prepare an intermediate dilution or increase the preparation volume. Dilution cannot produce a concentration above the stock.
TE Buffer (pH 8.0) — Recommended for Stocks
Sterile Water — For Working PCR Solutions
Sequence calculations: assumptions and limits
Sequence properties accept unmodified single-stranded DNA (A, C, G, T) written 5′ to 3′. Spaces and line breaks are ignored; RNA, ambiguous bases, FASTA headers, and modification annotations are not supported. MW assumes unmodified DNA with hydroxyl termini; use the supplier’s MW for modified oligos.
Wallace Tm = 2 × (A + T) + 4 × (G + C) is a rough composition-based estimate. It does not account for salt, Mg²⁺, primer concentration, mismatches, or secondary structure. Do not use it alone to choose an annealing temperature.
Frequently Asked Questions (FAQ)
Why do lyophilized primers need centrifugation before opening?
Briefly spin the closed tube to collect material from the cap and walls before opening. Use the supplier’s recommended centrifugation and mixing instructions.
What is the standard primer concentration for PCR reactions?
Most PCR protocols use a final primer concentration of 0.2 µM to 0.5 µM in the reaction mix. Preparing a 10 µM working stock makes pipetting easy (e.g. 1 µL into a 20–50 µL reaction).
How should reconstituted primers be stored?
For long-term storage of standard DNA oligos, IDT recommends −20°C, including for oligos in suitable TE buffer or nuclease-free water. Follow the supplier’s guidance for modified oligos; aliquots can help reduce handling and contamination.
How do I calculate volume if my tube label only shows OD260?
Use the supplier’s delivered nmol value if available. Otherwise use its sequence-specific extinction coefficient for OD260, or its mass and molecular weight. A universal OD-to-mass factor is only approximate for short oligos; this calculator accepts nmol, not raw absorbance.
Related calculators
Continue with dilution planning or buffer preparation using these CodonTable calculators.