Paste one unmodified DNA oligo in the 5′ to 3′ direction. The analyzer calculates nearest-neighbor melting temperature, GC content, molecular weight, A260 properties, and contiguous hairpin and self-dimer matches. Add a comparison oligo to check a primer pair for heterodimer complementarity under the same concentration and salt conditions.
For example, analyze this 28 nt primer at the default 250 nM total strand concentration and 50 mM Na+:
At the default conditions, their Tm values are 54.8 °C and 55.1 °C. The strongest heterodimer alignment has a 3 bp contiguous run at a 3′ end, so the screen marks it for review.
Input
Input
Accepted value
Limits
Behavior
Primary oligo
One DNA sequence using A, C, G, and T
8 to 120 nt
Required. Enter the strand in the 5′ to 3′ direction.
Comparison oligo
One DNA sequence using A, C, G, and T
8 to 120 nt
Optional. Adds a primer-pair heterodimer screen.
Text
Raw sequence or one FASTA record
Whitespace is ignored
Lowercase bases are converted to uppercase.
File
.txt, .fasta, .fa, .fas, or .seq
Up to 1 MB per input
Each input must contain one oligo.
Ambiguous bases such as N, RNA U, modifications, mixed-base notation, multiple FASTA records, and punctuation are rejected. Convert an RNA sequence to DNA first if U should be treated as T. The calculator does not infer or approximate modified-base chemistry.
Settings
Setting
What it controls
Allowed values
Default
Total strand concentration (nM)
Total concentration of both strands in the non-self-complementary duplex model. Tm uses the standard Ct/4 concentration term.
1 to 100,000 nM
250 nM
Na+ concentration (mM)
Monovalent sodium concentration used in the SantaLucia entropy correction.
1 to 1,000 mM
50 mM
Minimum hairpin loop (nt)
Smallest unpaired loop allowed between two contiguous complementary stem arms.
3 to 30 nt
3 nt
The Tm model does not include Mg2+, K+, Tris, dNTP binding, DMSO, formamide, mismatches, dangling ends, or chemical modifications. Use conditions that reflect the monovalent component of the intended experiment, and treat a calculated Tm as a model estimate rather than a measured transition temperature.
Results
Result
Meaning
Nearest-neighbor Tm
DNA/DNA duplex melting temperature from the Allawi and SantaLucia nearest-neighbor parameters with the SantaLucia 1998 monovalent-salt entropy correction.
GC content and Base counts
Fraction and count of G and C, plus counts for all four DNA bases.
Molecular weight
Average molecular mass of unmodified single-stranded DNA with 5′ and 3′ hydroxyl ends, in g/mol.
Extinction coefficient
Base-composition estimate at 260 nm, in M^-1 cm^-1.
nmol per OD260 and µg per OD260
Amount corresponding to one absorbance unit in a 1 cm path length, derived from the extinction coefficient and molecular weight.
Reverse complement
Reverse-complement sequence in the 5′ to 3′ direction. For a dedicated transform, use the reverse complement tool.
Primer checks
Practical review flags for 40% to 60% GC, 55 °C to 65 °C Tm, homopolymer length, and the final five-base GC clamp. These are common design ranges, not universal pass or fail rules.
Hairpin screen
Up to five gapless, contiguous Watson-Crick stems with dot-bracket notation, stem length, loop length, GC pairs, and 3′ involvement.
Self-dimer screen
Up to five antiparallel self-alignments ranked by 3′ complementarity, longest contiguous run, and total paired bases.
Primer-pair heterodimer screen
The same antiparallel screen between the primary and comparison oligos.
Thermodynamic terms
Duplex ΔH, ΔS, and salt-adjusted ΔS used in the Tm calculation.
CSV export contains the primary scalar properties for each oligo. Full JSON also preserves thermodynamic terms, checks, warnings, hairpin candidates, dimer alignments, and method labels.
Here, ΔH is in kcal/mol, ΔS is in cal/(mol·K), R is 1.987 cal/(mol·K), [Na+] is in mol/L, and Ct is the total strand concentration in mol/L. The implementation is checked against independent Biopython DNA_NN3 fixtures at the same concentrations.
What do the hairpin and primer-dimer results mean?
The structure screens answer a narrow, reproducible question: where can two uninterrupted DNA segments form Watson-Crick pairs in an antiparallel orientation? They report the exact alignment, the longest run, and whether a run reaches a 3′ end. Hairpins also enforce the configured minimum loop and show dot-bracket notation.
Signal labels are screening aids:
Low signal: a 2 to 4 bp contiguous match without a long 3′ run.
Review: a 5 to 6 bp run, or at least 3 contiguous pairs at a 3′ end.
Strong signal: a run of at least 7 bp, at least 4 contiguous pairs at a 3′ end, or a hairpin stem of at least 6 bp. A 4 bp hairpin involving the 3′ end is also marked strong.
These labels do not represent free energy. The screen does not calculate ΔG, Tm for a folded structure, bulges, mismatches, internal loops, coaxial stacking, or competing structure ensembles. OligoCalc's current documentation also describes its historical homology-based hairpin and dimer checks as deprecated, while Primer3 provides a stronger thermodynamic model for consequential secondary-structure decisions. Vendor calculations can differ because their conditions, modifications, and parameter sets differ.
Molecular weight and A260 assumptions
Molecular weight uses average residue masses for unmodified DNA and subtracts 61.96 g/mol for a strand with 5′ and 3′ hydroxyl ends, matching the convention documented by OligoCalc. A 5′ phosphate, dye, quencher, linker, phosphorothioate, or other modification changes the mass and may change absorbance.
The extinction coefficient is a base-composition estimate. It is useful for a consistent planning value, but sequence context and experimental calibration can matter. Cavaluzzi and Borer found that common base-composition and nearest-neighbor absorbance estimates can systematically differ from measured unpaired DNA values, as discussed in their revised UV extinction coefficient study.
Which oligo or primer tool should I use?
Goal
Tool
Review one primer or a primer pair for Tm, mass, A260 properties, hairpins, and dimers
Oligo analyzer
Calculate composition and sliding-window GC across longer DNA or RNA records
Yes. It calculates a salt-adjusted nearest-neighbor DNA/DNA Tm from total strand concentration and Na+ concentration. It is not a Wallace-rule estimate.
Can I check two primers for primer dimers?
Yes. Put the forward primer in Primary oligo and the second primer, written 5′ to 3′, in Comparison oligo. The heterodimer screen aligns them antiparallel and ranks 3′ complementarity first.
Does the hairpin checker calculate ΔG?
No. It finds gapless contiguous Watson-Crick stems and reports stem length, loop length, GC pairs, 3′ involvement, and dot-bracket notation. Use a thermodynamic secondary-structure method when ΔG or structure ensembles affect the decision.
Why does my Tm differ from IDT OligoAnalyzer?
IDT OligoAnalyzer can account for additional ions, target types, and ordered modifications. Results also change with concentration conventions and thermodynamic parameter sets. Match the stated conditions before comparing values.
Can I enter RNA, degenerate bases, or modified oligos?
No. The accepted alphabet is unambiguous DNA A, C, G, and T. Rejecting unsupported chemistry prevents a precise-looking result based on the wrong constants.
What primer Tm and GC content should I target?
The result view flags 55 °C to 65 °C Tm and 40% to 60% GC as common PCR primer ranges. Assay chemistry, polymerase, amplicon, cycling protocol, and primer pairing determine the final target, so these ranges are review aids rather than universal requirements.
