Oligo Calculator

An oligo calculator (or oligocalc) calculates the physical and chemical properties of a short DNA or RNA sequence: melting temperature (Tm), molecular weight, GC percentage, extinction coefficient at 260 nm, and resuspension volume. Researchers use these values when designing PCR primers, probes, CRISPR guide RNAs, siRNAs and sequencing primers.

For oligonucleotides between 8 and 40 nt, the nearest-neighbor thermodynamic method is the most accurate. It accounts for stacking interactions between adjacent base pairs, not just GC count. The salt-adjusted formula is preferred for longer sequences. The Wallace rule (Tm = 2AT + 4GC) is simplest but least accurate — use only for very short oligos (<13 nt) or quick estimates.

Enter the nmol amount printed on your oligo vial (actual yield — not the synthesis scale ordered). Enter your desired stock concentration — 100 µM is standard for PCR primers. The calculator divides: Volume (µL) = nmol ÷ µM. For example, 10 nmol ÷ 100 µM = 100 µL of TE buffer or nuclease-free water.

Stock concentration (usually 100 µM) is the concentrated master solution stored long-term at −20°C. Working concentration (usually 10 µM) is a diluted aliquot kept at 4°C for regular use. This protects your stock from repeated freeze-thaw cycles. Final primer concentration in a PCR reaction is typically 200–500 nM per primer.

Optimal GC content for PCR primers is 40–60%. Below 40%, the Tm will be low, increasing non-specific amplification. Above 60%, secondary structures like hairpins become more likely. End the primer with a GC clamp — 1–2 G or C bases at the 3′ end — which improves binding efficiency.

For ssDNA: MW = (A × 313.21) + (T × 304.2) + (C × 289.18) + (G × 329.21) − 61.96. The −61.96 accounts for the loss of water during strand formation. For dsDNA, calculate both strands and sum them. For RNA, replace T (304.2) with U (306.17) and add 159.0 for the 5′ triphosphate if calculating a transcript.