Oligonucleotide Tm Calculator

Chemistry

Oligo sequence (5′→3′)

DNA · 19 nt · GC 57.9 %

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Method and conditions

Custom

Method

Nearest-neighbourGC formulaWallace

Duplex type

Nearest-neighbour table

Salt correction

Na⁺ (mM)

K⁺ (mM)

Tris, total (mM)

Mg²⁺ (mM)

dNTPs, total (mM)

Oligo concentration

Complementary strand

DMSO (%)

Formamide (%)

Polymerase vendors' calculators use their own buffer models and offsets, so their numbers will differ.

55.4 °C

melting temperature

Nearest-neighbour · SantaLucia 1998 · Owczarzy 2008 · 50 mM Na⁺ · 250 nM

Thermodynamics

ΔH°

−148.3 kcal/mol

ΔS° (1 M Na⁺)

−400.8 cal/(K·mol)

ΔS effective

−419.8 cal/(K·mol)

ΔG°37 at 1 M Na⁺

−23.99 kcal/mol

ΔG°37 at your conditions

−18.09 kcal/mol

k (effective strand concentration)

125.0 nM

Owczarzy 2008, monovalent-dominated regime: no free Mg²⁺

Oligo properties

Length

19 nt

GC content

57.9 %

Base counts

A 5 · C 2 · G 9 · T 3

Formula

C₁₈₈H₂₃₂N₈₂O₁₁₀P₁₈

Average MW

5957.92 g/mol

Monoisotopic mass

5955.0358 Da

ε260 (single strand)

197,100 M⁻¹ cm⁻¹

Reverse complement

GACTCCGACCTTCACCTTC

5′ end (changes the mass, not the Tm)

3′ end

To turn an A260 reading into a concentration, use the Nucleic Acid Concentration Calculator. For ESI ions of this oligo, use the Mass Spectrometry Adduct Calculator with the formula above, or paste the formula into the Molar Mass Calculator.

Primer checks

Rules of thumb. Your polymerase and assay may tolerate more.

OK
Length
19 nt, inside 18–30 nt.
OK
GC content
57.9 %, inside 40–60 %.
OK
Tm (Nearest-neighbour · SantaLucia 1998)
55.4 °C, inside 52–65 °C.
OK
GC clamp
3 G/C in the last 5 bases.
OK
Runs
no runs of 4 or more identical bases or dinucleotide repeats.
Info
3′ pentamer ΔG°37
GAGTC: −2.06 kcal/mol. Less negative means a weaker 3′ end.
Scale: all 1,024 pentamers at your salt, in kcal/mol.
OK
Hairpin
no stem of 4 bp or more with a loop of 3 nt or more.
OK
Self-dimer
none of 4 bp or more

Melting curve

Percent of the limiting strand in duplex against temperature.

Oligo

At 50.0 °C: 89.9 % in duplex

Two-state model: each strand is either fully paired or fully single-stranded. Hover, tap or use the arrow keys to move the temperature cursor.

Duplex and stability map

Each bar is the ΔG°37 of one stacking step at 1 M Na⁺; the table shows where the Tm comes from.

Hover or focus a bar to read that step. Top: oligo (5′→3′). Bottom: complement (3′→5′).

ΔH in kcal/mol, ΔS in cal/(K·mol).
Term	ΔH	ΔS	Tm
5′ terminal pair G·C	0.10	−2.80
3′ terminal pair G·C	0.10	−2.80
Sum of 18 nearest-neighbour steps	−148.50	−395.20
Total at 1 M Na⁺	−148.30	−400.80	69.83 °C
Salt correction (Owczarzy 2008)			−14.46 °C
Result at your conditions	−148.30	−419.82	55.38 °C

Method comparison

The same sequence and conditions under every method.

★ marks the method you selected. Calculators disagree mostly because they use different methods and assume different salt and oligo concentrations, not because one is broken.

Tm versus conditions

One setting varies; everything else stays as you set it.

Mg²⁺Na⁺OligoDMSO

1.5 mM Mg²⁺ raises this primer's Tm by 6.0 °C.

Everything else stays at your current settings. A filled-ring dot marks the current value.

Oligonucleotide Tm Calculator – Melting Temperature of Primers and Probes

Paste a primer, probe or short oligo and this oligonucleotide Tm calculator returns its melting temperature under your own buffer: Na⁺, K⁺, Tris, Mg²⁺, dNTPs, oligo concentration, DMSO and formamide. It also shows the thermodynamics behind the number, a melting curve, the molecular weight and ε260, and the usual primer-design checks. Everything is calculated in your browser.

What Tm means

In the two-state model each strand is either fully paired or fully single-stranded. The Tm is the temperature at which half of the limiting strand is paired. Because melting is a binding equilibrium, Tm is not a property of the sequence alone: doubling the strand concentration raises it, and so does more salt.

The nearest-neighbour idea

A duplex is stabilised by the stacking of neighbouring base pairs, not by counting G and C. The nearest-neighbour method adds a tabulated ΔH° and ΔS° for each of the N−1 dinucleotide steps, plus initiation and terminal terms, then solves for the temperature where the duplex is half melted:

Tm = 1000·ΔH° / (ΔS° + R·ln k) − 273.15

Here R = 1.987 cal/(K·mol) and k is the effective strand concentration. The tool offers SantaLucia 1998, SantaLucia & Hicks 2004 and Breslauer 1986 for DNA, Xia 1998 for RNA and Sugimoto 1995 for RNA/DNA hybrids.

Wallace and GC-content formulas

The Wallace rule, 2·(A+T) + 4·(G+C), is a quick estimate for 14–20 nt oligos. The GC formulas (Basic, Marmur–Schildkraut, von Ahsen, QuikChange) add length and, for some, salt. They are fine for a rough check or for QuikChange mutagenesis primers, but they ignore the stacking sequence, so they can differ from a nearest-neighbour value by several degrees.

Salt, Mg²⁺ and dNTPs

Cations screen the phosphate backbone. The default Owczarzy 2008 correction treats monovalent ions and Mg²⁺ separately, because Mg²⁺ stabilises a duplex far more per mole. dNTPs bind Mg²⁺ almost one to one, so only the free Mg²⁺ counts. A typical PCR buffer with 1.5 mM Mg²⁺ and 0.8 mM dNTPs leaves about 0.7 mM free, and raises a primer's Tm by several degrees over a plain 50 mM Na⁺ buffer.

DMSO and formamide

Both destabilise the duplex. The tool subtracts 0.75 °C per percent DMSO and 0.65 °C per percent formamide, and you can edit those factors.

Why calculators disagree

Two websites can give the same primer Tms 10 °C apart. The causes are the method, the parameter table, the salt correction and the assumed concentrations. The method comparison strip puts them side by side so you can see which assumption moves the number.

Picking an annealing temperature

Start 3–5 °C below the lower primer Tm in your real reaction buffer, keep the two primers within a few degrees of each other, and avoid primers whose 3′ ends pair with each other or fold into a hairpin. The melting curve shows why this works: a few degrees below Tm the primer is mostly bound, while a few degrees above it is mostly free.

Degenerate primers

A primer with codes such as N, R, Y or M is a mixture of every concrete sequence it allows. The calculator expands up to 4,096 variants and reports the lowest, highest and mean Tm. Choose the annealing temperature for the lowest one, because the weakest variant limits the reaction.

A Tm is a model prediction

The value depends on the model and on buffer conditions the calculator can only approximate. Treat it as a starting point and confirm the annealing temperature with a gradient PCR, which is the ground truth.

Is the Oligonucleotide Tm Calculator free?

Yes, Oligonucleotide Tm Calculator is totally free :)

Can I use the Oligonucleotide Tm Calculator offline?

Yes, you can install the webapp as PWA.

Is it safe to use Oligonucleotide Tm Calculator?

Yes, any data related to Oligonucleotide Tm Calculator only stored in your browser (if storage required). You can simply clear browser cache to clear all the stored data. We do not store any data on server.

What is the melting temperature (Tm) of a primer?

Tm is the temperature at which half of the primer molecules are paired with their complementary strand and half are single-stranded. It is not a fixed property of the sequence: it moves with the oligo concentration, the salt, Mg²⁺ and dNTPs in the buffer and with additives such as DMSO. That is why this calculator asks for your actual reaction conditions.

Why do different Tm calculators give different results for the same primer?

They differ in the method (nearest-neighbour or a base-counting formula), in the published parameter table, in the salt correction and in the buffer and primer concentration they assume. Differences of 5 to 10 °C are common between a simple GC formula and a nearest-neighbour calculation. The comparison strip shows every method side by side for your sequence and conditions.

How do Mg²⁺ and dNTPs change primer Tm?

Divalent Mg²⁺ stabilises the duplex far more strongly than Na⁺ or K⁺, so 1.5 mM Mg²⁺ typically raises a primer's Tm by several degrees. dNTPs bind Mg²⁺ almost one to one, so only the free Mg²⁺ (total Mg²⁺ minus dNTPs, roughly) counts. The Owczarzy 2008 correction models this free Mg²⁺ directly, which is why it is the default.

What annealing temperature should I use for PCR?

A common starting point is 3 to 5 °C below the lower Tm of the two primers, calculated under your real PCR buffer. Treat it as a first guess and refine it with a gradient PCR, because the polymerase, template and buffer all shift the optimum. Raising the temperature increases specificity, and lowering it increases yield.

How is Tm calculated for degenerate primers?

A degenerate primer is a mixture of every concrete sequence its ambiguity codes allow, for example 18 variants for 806R. The calculator expands all of them, runs the chosen method on each and reports the lowest, highest and mean Tm. For choosing an annealing temperature, use the lowest Tm, because the weakest variant limits it.

Why does oligo concentration affect Tm?

Melting is a two-molecule equilibrium for a duplex of two strands, so a higher strand concentration pushes the equilibrium toward the duplex and raises the Tm. In the nearest-neighbour equation it appears as R·ln(k). Going from 25 nM to 1 µM raises a typical primer's Tm by roughly 4 to 7 °C.