Oligo Resuspension & Dilution Calculator

Professional calculator for oligonucleotide preparation. Calculate resuspension volumes, C₁V₁=C₂V₂ dilutions, and pool-specific concentrations with instant printable protocols.

Free & instant - No registration required
IDT/Twist compatible - Works with all major vendors
Validated formulas - Industry-standard calculations

When to Use Each Calculator

1Resuspension Calculator

Use for: Initial reconstitution of lyophilized oligos from vendor. Input received nmol, get buffer volume for target stock (typically 100 µM).

2Dilution Calculator

Use for: Preparing working solutions from stocks using C₁V₁=C₂V₂. Common: 100 µM stock → 10 µM working solution for PCR.

3Pool-Specific Calculator

Use for: CRISPR libraries and oligo pools requiring equimolar representation. Calculate per-oligo concentration (standard: 0.5-2 nM each).

Input Parameters

Amount stated on vendor tube/datasheet

Common: 100 µM for primers, 10 µM for working stocks

If provided, will calculate per-oligo concentration

Results

No results yet

Enter values and click "Calculate Volume"

Why Accurate Oligo Dilution Matters

When you receive synthesized oligonucleotides from a vendor, they typically arrive as a lyophilized (freeze-dried) pellet or in a concentrated stock solution. Before using them in experiments, you need to resuspend (if lyophilized) and dilute them to the correct working concentration. Incorrect oligo concentration is one of the most common causes of PCR failure, qPCR variability, and CRISPR library unevenness.

Our Dilution Calculator handles two common scenarios: (1) Resuspension — calculating the volume of buffer to add to a lyophilized oligo to achieve a desired stock concentration (typically 100 µM), and (2) Serial dilution — using the C1V1 = C2V2 formula to dilute from stock concentration to working concentration (typically 10 µM for PCR primers or 0.1-1 µM for oligo pools).

For oligo pools, concentration calculation is more nuanced because the pool contains many different sequences. The calculator accounts for the total nmol of the pool, the number of unique sequences, and provides per-sequence concentration to help you achieve adequate representation. This is critical for CRISPR library screening where each sgRNA must be represented at a minimum copy number.

How to Use the Dilution Calculator

  1. Choose your mode: Resuspension (lyophilized oligo to stock solution) or Dilution (stock to working concentration).
  2. For Resuspension: enter the amount of oligo (in nmol or OD260, as reported by vendor) and desired stock concentration (e.g., 100 µM). The calculator shows the volume of TE buffer or nuclease-free water to add.
  3. For Dilution: enter the stock concentration (C1), desired final concentration (C2), and final volume (V2). The calculator shows the volume of stock (V1) and buffer to add.
  4. For oligo pools: enter total pool amount, number of sequences, and desired per-sequence concentration. The calculator provides total resuspension volume and per-sequence representation.
  5. Review the generated step-by-step protocol — you can print or save it for your lab notebook.

Frequently Asked Questions

What buffer should I use to resuspend oligonucleotides?
For most applications, use TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) for long-term storage, or nuclease-free water for immediate use. TE buffer provides pH buffering and the EDTA chelates divalent cations that could catalyze degradation. Avoid using DEPC-treated water for DNA oligos (DEPC can chemically modify adenine bases). For oligos with special modifications (e.g., locked nucleic acids), follow the vendor's specific resuspension recommendations.
How long can I store resuspended oligos?
Stock solutions (100 µM in TE buffer) can be stored at -20°C for months to years with minimal degradation. Avoid repeated freeze-thaw cycles — aliquot into single-use volumes. Working dilutions (1-10 µM) should be prepared fresh or stored at -20°C for no more than a few weeks. At 4°C, dilute oligo solutions degrade within days due to nuclease contamination and hydrolysis. Never store oligos in water at room temperature.
What is the C1V1 = C2V2 formula?
C1V1 = C2V2 (also written as M1V1 = M2V2) is the dilution equation where C1 = initial concentration, V1 = volume of stock solution needed, C2 = desired final concentration, and V2 = desired final volume. To find V1 (how much stock to use): V1 = (C2 × V2) / C1. For example, to make 100 µL of 10 µM primer from a 100 µM stock: V1 = (10 × 100) / 100 = 10 µL of stock + 90 µL of buffer.
How do I calculate concentration for oligo pools?
For oligo pools, the vendor reports total pool amount (e.g., 100 pmol). Per-sequence concentration = (total pool amount) / (number of unique sequences × resuspension volume). For a pool of 1,000 sequences at 100 pmol total, resuspended in 100 µL: per-sequence concentration = 100 pmol / (1000 × 100 µL) = 0.001 µM = 1 nM per sequence. This per-sequence concentration determines your screening coverage in CRISPR experiments.
Why is my PCR failing even with correct primer concentration?
Even with correct molar concentration, PCR can fail if: (1) the original quantification was inaccurate — NanoDrop readings are affected by salts and contaminants, so verify with a fluorometric method (Qubit) for critical applications; (2) primers degraded during storage — check storage conditions; (3) primers have strong secondary structures that sequester the functional primer fraction — check with our Secondary Structure Predictor; (4) primer concentration is correct but Mg²⁺ or dNTP concentrations are not optimized for your specific buffer system.

Related Tools

Molecular Weight Calculator

Calculate MW and extinction coefficient needed for accurate concentration determination.

Tm Calculator

After preparing your primers, verify Tm to set the correct PCR annealing temperature.

Batch Sequence QC

Quality-check your oligo pool sequences before resuspension and library preparation.

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Validate PCR Primers Before OrderingPre-Order Oligo Pool QCUser Guide