{"id":66885,"date":"2026-10-13T10:06:11","date_gmt":"2026-10-13T07:06:11","guid":{"rendered":"https:\/\/wpcalc.com\/en\/?p=66885"},"modified":"2026-10-13T10:06:11","modified_gmt":"2026-10-13T07:06:11","slug":"dna-melting-temperature-calculator","status":"publish","type":"post","link":"https:\/\/wpcalc.com\/en\/chemistry\/dna-melting-temperature-calculator\/","title":{"rendered":"DNA Melting Temperature Calculator"},"content":{"rendered":"<section class=\"not-prose calculator-box\" aria-labelledby=\"calculator-title\"><div class=\"flex items-baseline sm:items-center justify-between gap-2 sm:gap-3\"><div class=\"flex flex-col sm:flex-row sm:items-center gap-2\"><span class=\"icon icon-calculator text-primary-700! text-base! dark:text-primary-300!\" aria-hidden=\"true\"><\/span><h2 id=\"calculator-title\" class=\"text-lg font-display font-bold\">Estimate the Melting Temperature of a DNA Oligonucleotide<\/h2><\/div><div class=\"calcdash-favorite group\">\n  <button type=\"button\" class=\"favorite\" id=\"favorite\" data-favorite-id=\"66885\" data-favorite-title=\"DNA Melting Temperature\" data-favorite-url=\"https:\/\/wpcalc.com\/en\/chemistry\/dna-melting-temperature-calculator\/\" data-favorite-excerpt=\"Estimate the melting temperature (Tm) of a DNA oligonucleotide from its sequence and salt concentration. Enter one strand (A, C, G, T) and the Na\u207a concentration to get the Tm and GC content.\" aria-label=\"Add to Favorites\" data-favorite-icon=\"icon icon-biology\">\n    <span class=\"icon icon-shape-star-empty\" aria-hidden=\"true\"><\/span>\n  <\/button>\n  <div class=\"calcdash-favorite__tip\" aria-hidden=\"true\">\n   <span class=\"favorite-tooltip\" id=\"favorite-tooltip\"><\/span>\n  <\/div>\n  <span class=\"sr-only\" role=\"status\" id=\"favorite-status\"><\/span>\n<\/div><\/div><form action=\"https:\/\/wpcalc.com\/en\/chemistry\/dna-melting-temperature-calculator\/\" method=\"POST\" class=\"calculator\" id=\"calculator-66885\" data-post=\"66885\"><fieldset class=\"fieldset-input is-1\"><legend class=\"sr-only\">Input Fields<\/legend><div class=\"field is-single\" id=\"input-1\"><label for=\"field-1\">DNA Sequence (5\u2032\u21923\u2032)<\/label><textarea id=\"field-1\" name=\"dna_sequence_53\">ATGCATGCGATTACA<\/textarea><\/div><div class=\"field has-term\" id=\"input-2\"><label for=\"field-2\">Salt (Na\u207a) Concentration<\/label><div class=\"term\">[Na\u207a]<\/div>  <div class=\"absolute bottom-0 left-16 top-8 w-2 h-0.5 bg-blue-500\"><\/div><input type=\"text\" name=\"salt_na_concentration\" id=\"field-2\" aria-describedby=\"calc-unit-2 calc-info-2\" data-number inputmode=\"decimal\" autocomplete=\"off\" spellcheck=\"false\" placeholder=\"e.g. 50\" min=\"0\" value=\"50\"\/><div class=\"unit\" id=\"calc-unit-2\">mM<\/div><small id=\"calc-info-2\">Total monovalent salt concentration. Used by the salt-adjusted formula for sequences of 14 bases or longer; the Wallace rule for shorter oligos does not use it.<\/small><\/div><\/fieldset><div class=\"buttons\"><button type=\"submit\" data-text=\"Re-Calculate\" id=\"calculate-button\" data-post=\"66885\">Calculate<\/button><button type=\"reset\">Reset<\/button><\/div><div class=\"field is-checkbox hidden!\" id=\"field-auto-calc\"><input type=\"checkbox\" id=\"auto-calc\"><label for=\"auto-calc\">Calculate automatically<\/label><small>If enabled, the result will update automatically when you change any value.<\/small><\/div><div class=\"fieldset-result is-hidden\" aria-labelledby=\"result-title\" aria-live=\"polite\" role=\"region\"> <h3 class=\"result-title bg-gradient-to-r from-primary-50 to-gray-50 dark:from-primary-900 dark:to-gray-800\" id=\"result-title\"><span class=\"icon icon-s-pulse\" aria-hidden=\"true\"><\/span> Melting Temperature and GC Content<\/h3><div class=\"result-box\"><div class=\"field-result\" id=\"output-1\"><span class=\"field-title\"><span>Melting Temperature (Tm)<\/span><span class=\"result-term\">Tm<\/span><\/span><span class=\"field-value\" id=\"result-1\" data-unit=\"\u00b0C\"><\/span><button type=\"button\" class=\"copy-result\" data-tooltip=\"Copy Result\" aria-label=\"Copy Result\"><span class=\"copy-icon icon icon-document-copy\" aria-hidden=\"true\"><\/span><\/button><\/div><div class=\"field-result\" id=\"output-2\"><span class=\"field-title\"><span>GC Content<\/span><span class=\"result-term\">GC<\/span><\/span><span class=\"field-value\" id=\"result-2\" data-unit=\"%\"><\/span><button type=\"button\" class=\"copy-result\" data-tooltip=\"Copy Result\" aria-label=\"Copy Result\"><span class=\"copy-icon icon icon-document-copy\" aria-hidden=\"true\"><\/span><\/button><\/div><div class=\"field-result is-column\" id=\"output-3\"><span class=\"field-title\"><span>Calculation Details<\/span><\/span><span class=\"field-value\" id=\"result-3\"><\/span><button type=\"button\" class=\"copy-result\" data-tooltip=\"Copy Result\" aria-label=\"Copy Result\"><span class=\"copy-icon icon icon-document-copy\" aria-hidden=\"true\"><\/span><\/button><\/div><div class=\"field-result is-column\" id=\"output-4\"><span class=\"field-title\"><span>About the Formulas and Assumptions<\/span><\/span><span class=\"field-value\" id=\"result-4\"><\/span><button type=\"button\" class=\"copy-result\" data-tooltip=\"Copy Result\" aria-label=\"Copy Result\"><span class=\"copy-icon icon icon-document-copy\" aria-hidden=\"true\"><\/span><\/button><\/div><\/div><div class=\"result-actions\"><button type=\"button\" class=\"copy-link\"><svg viewBox=\"0 0 20 20\" fill=\"currentColor\" aria-hidden=\"true\"><path d=\"M12.232 4.232a2.5 2.5 0 0 1 3.536 3.536l-1.225 1.224a.75.75 0 0 0 1.061 1.06l1.224-1.224a4 4 0 0 0-5.656-5.656l-3 3a4 4 0 0 0 .225 5.865.75.75 0 0 0 .977-1.138 2.5 2.5 0 0 1-.142-3.667l3-3Z\"\/><path d=\"M11.603 7.963a.75.75 0 0 0-.977 1.138 2.5 2.5 0 0 1 .142 3.667l-3 3a2.5 2.5 0 0 1-3.536-3.536l1.225-1.224a.75.75 0 0 0-1.061-1.06l-1.224 1.224a4 4 0 1 0 5.656 5.656l3-3a4 4 0 0 0-.225-5.865Z\"\/><\/svg><span class=\"copy-link__text\">Copy link to this calculation<\/span><\/button><span class=\"sr-only\" role=\"status\" id=\"copy-link-status\"><\/span><\/div><\/div><a href=\"#respond\" class=\"hidden transition-opacity duration-300 opacity-0 w-50 text-sm justify-center items-center gap-2 px-4 py-2 rounded bg-gray-200 text-gray-700 hover:bg-gray-300\" id=\"leave-comment\"><span class=\"icon icon-comments\" aria-hidden=\"true\"><\/span>Leave a Comment<\/a><\/form><\/section><section id=\"calc-reactions\" class=\"not-prose hidden my-12 bg-gradient-to-r from-primary-50 to-gray-50 border border-indigo-100 rounded-xl px-6 py-4 shadow-sm\" data-post=\"66885\" aria-live=\"polite\" aria-labelledby=\"calc-reactions-title\"><p id=\"calc-reactions-title\" class=\"text-sm text-gray-500 text-center mb-2\">How did this result make you feel?<\/p><div role=\"group\" aria-labelledby=\"calc-reactions-title\" class=\"grid grid-cols-3 sm:flex sm:flex-row sm:justify-around gap-4 sm:items-center sm:flex-wrap\"><button type=\"button\" aria-pressed=\"false\" class=\"reaction-btn flex flex-col items-center gap-1 text-sm text-gray-700 hover:text-primary-600 cursor-pointer transition-transform hover:scale-105\" data-reaction=\"like\"><span class=\"reaction-count\" data-reaction-count=\"like\">0<\/span><span class=\"reaction text-3xl\" aria-hidden=\"true\">\ud83d\ude00 <\/span><span class=\"reaction-description font-medium\">Like <\/span><\/button><button type=\"button\" aria-pressed=\"false\" class=\"reaction-btn flex flex-col items-center gap-1 text-sm text-gray-700 hover:text-primary-600 cursor-pointer transition-transform hover:scale-105\" data-reaction=\"helpful\"><span class=\"reaction-count\" data-reaction-count=\"helpful\">0<\/span><span class=\"reaction text-3xl\" aria-hidden=\"true\">\ud83d\udca1 <\/span><span class=\"reaction-description font-medium\">Helpful <\/span><\/button><button type=\"button\" aria-pressed=\"false\" class=\"reaction-btn flex flex-col items-center gap-1 text-sm text-gray-700 hover:text-primary-600 cursor-pointer transition-transform hover:scale-105\" data-reaction=\"confused\"><span class=\"reaction-count\" data-reaction-count=\"confused\">0<\/span><span class=\"reaction text-3xl\" aria-hidden=\"true\">\ud83d\ude15 <\/span><span class=\"reaction-description font-medium\">Confused <\/span><\/button><button type=\"button\" aria-pressed=\"false\" class=\"reaction-btn flex flex-col items-center gap-1 text-sm text-gray-700 hover:text-primary-600 cursor-pointer transition-transform hover:scale-105\" data-reaction=\"disappointed\"><span class=\"reaction-count\" data-reaction-count=\"disappointed\">0<\/span><span class=\"reaction text-3xl\" aria-hidden=\"true\">\ud83d\ude1e <\/span><span class=\"reaction-description font-medium\">Disappointed <\/span><\/button><button type=\"button\" aria-pressed=\"false\" class=\"reaction-btn flex flex-col items-center gap-1 text-sm text-gray-700 hover:text-primary-600 cursor-pointer transition-transform hover:scale-105\" data-reaction=\"inaccurate\"><span class=\"reaction-count\" data-reaction-count=\"inaccurate\">0<\/span><span class=\"reaction text-3xl\" aria-hidden=\"true\">\u274c <\/span><span class=\"reaction-description font-medium\">Inaccurate <\/span><\/button><\/div><div id=\"reaction-message\" class=\"hidden mt-8 rounded-md border border-primary-100 bg-white\/50 backdrop-blur-sm px-4 py-2 text-sm text-center shadow-sm\"><\/div><\/section><div class=\"calcdash-ad not-prose calcdash-ad--calc_result calcdash-ad--in-article\"><ins class=\"adsbygoogle\" style=\"display:block\" data-ad-client=\"ca-pub-1721569815777345\" data-ad-slot=\"6317458308\" data-ad-layout=\"in-article\" data-ad-format=\"fluid\"><\/ins><script>(adsbygoogle = window.adsbygoogle || []).push({});<\/script><\/div>\n<style id=\"wpcoderpro-inline-1\">.formula-box {\r\n\tborder-bottom: none !important;\r\n}<\/style><section class=\"formula-box\" aria-labelledby=\"formula-title\">\r\n\t<div id=\"formula-title\" class=\"not-prose font-display mb-4\"><span class=\"icon icon-formula\" aria-hidden=\"true\"><\/span><h2>DNA Melting Temperature Formula<\/h2><\/div>\r\n\t<figure class=\"not-prose formula\">\r\n\t\t<figcaption class=\"formula-title\">Formula<\/figcaption>\r\n\t\t<div class=\"text-base text-gray-800\" id=\"formula\">\n\\[ T_m = 2\\,(A+T) + 4\\,(G+C) \\\\ T_m = 100.5 + 41\\,\\frac{G+C}{n} - \\frac{820}{n} + 16.6\\,\\log_{10}[\\mathrm{Na^+}] \\]\n<\/div>\r\n\t<\/figure>\r\n\t<\/p>\n<p><strong>Where:<\/strong><\/p>\n<ul>\n<li>\\(A, T, G, C\\) \u2014 number of adenine, thymine, guanine and cytosine bases in the sequence<\/li>\n<li>\\(n\\) \u2014 total number of bases (sequence length)<\/li>\n<li>\\([\\mathrm{Na^+}]\\) \u2014 total monovalent salt concentration in mol\/L<\/li>\n<\/ul>\n<p>The first equation is the Wallace rule, used for short oligonucleotides up to 13 bases; the second is the salt-adjusted formula (Howley et al., 1979), applied automatically from 14 bases onward. The calculator picks the appropriate formula for you.<\/p>\n<p>\r\n<\/section>\n<style id=\"wpcoderpro-inline-459\">.calculation-box {\r\n\tborder-bottom: none !important;\r\n}<\/style><section id=\"calculation\" class=\"calculation-box\" aria-labelledby=\"calculation-title\">\r\n\t<div id=\"calculation-title\" class=\"not-prose font-display\"><span class=\"icon icon-unordered-list\" aria-hidden=\"true\"><\/span><h2>DNA Melting Temperature \u2013 Calculation Example<\/h2><\/div>\r\n\r\n<\/p>\n<p><strong>Example:<\/strong> a 15-base oligonucleotide with the sequence ATGCATGCGATTACA in a buffer containing 50 mM Na\u207a. The sequence contains 5 A, 4 T, 3 G and 3 C bases, so \\(G + C = 6\\), \\(A + T = 9\\) and \\(n = 15\\).<\/p>\n<p>\\[ [\\mathrm{Na^+}] = 50\\ \\mathrm{mM} = 0.05\\ \\mathrm{mol\/L}, \\quad \\log_{10}(0.05) = -1.301 \\]<br \/>\n\\[ T_m = 100.5 + 41 \\cdot \\frac{6}{15} - \\frac{820}{15} + 16.6 \\cdot (-1.301) \\]<br \/>\n\\[ T_m = 100.5 + 16.4 - 54.67 - 21.60 = 40.6\\ \\mathrm{\u00b0C} \\]<\/p>\n<p><strong>Result:<\/strong> the melting temperature is \\(T_m \\approx 40.6\\) \u00b0C and the GC content is 40.0%. The simple Wallace rule would give 2 \u00d7 9 + 4 \u00d7 6 = 42 \u00b0C, but it is not recommended for sequences of 14 bases and longer.<\/p>\n<p>\n\r\n<\/section>\n<h2>How to Use the Calculator<\/h2>\n<ol>\n<li>Type or paste your sequence into the <strong>DNA Sequence (5\u2032\u21923\u2032)<\/strong> field. Only the letters A, C, G and T are accepted; case, spaces and line breaks are ignored.<\/li>\n<li>Enter the total monovalent salt concentration in the <strong>Salt (Na\u207a) Concentration<\/strong> field, in mM (default 50 mM). This value is used only when the sequence has 14 bases or more.<\/li>\n<li>Read the <strong>Melting Temperature (Tm)<\/strong> in \u00b0C and the <strong>GC Content<\/strong> in percent in the results panel.<\/li>\n<li>Check <strong>Calculation Details<\/strong> to see the base composition, which formula was applied and the substituted numbers, so you can verify the result by hand.<\/li>\n<\/ol>\n<h2>What Is the Melting Temperature?<\/h2>\n<p>The melting temperature, or Tm, is the temperature at which half of the DNA duplexes in a solution are dissociated into single strands and half remain paired. It is a direct measure of duplex stability: the higher the Tm, the more heat is needed to separate the two strands. The Tm matters whenever two complementary DNA strands must find and bind each other reliably \u2014 designing PCR primers, choosing hybridisation probe conditions, or planning annealing temperatures for short oligos.<\/p>\n<p>Duplex stability comes from the base pairs themselves. A G\u2013C pair is held by three hydrogen bonds while an A\u2013T pair has only two, so GC-rich sequences melt at higher temperatures. In the Wallace rule this is expressed very directly: each A\u2013T pair adds roughly 2 \u00b0C to the Tm and each G\u2013C pair roughly 4 \u00b0C. The GC content reported alongside the Tm tells you how the sequence is composed \u2014 a quick sanity check, since GC content and Tm always rise and fall together.<\/p>\n<p>Salt also plays an important role. Sodium ions shield the negative charges on the phosphate backbone, so a higher Na\u207a concentration stabilises the duplex and raises the Tm. This is why the salt-adjusted formula includes the term \\(16.6\\,\\log_{10}[\\mathrm{Na^+}]\\): every tenfold increase in salt adds about 16.6 \u00b0C to the predicted Tm. When comparing oligos, always compare them at the same salt concentration, and remember that the salt concentration must be entered in mM \u2014 the conversion to mol\/L is done automatically.<\/p>\n<h2>Accuracy, Assumptions and Practical Tips<\/h2>\n<ul>\n<li><strong>Both formulas assume a perfect duplex:<\/strong> the sequence must hybridise to a fully complementary strand with no mismatches, bulges or gaps. Even a single mismatch can lower the true Tm by several degrees.<\/li>\n<li><strong>Wallace rule limits:<\/strong> it is a rough rule of thumb intended for roughly 4\u201313 bases near 50 mM Na\u207a. It ignores salt concentration and sequence length effects, so use it only for quick estimates on very short oligos.<\/li>\n<li><strong>Salt-adjusted formula limits:<\/strong> it accounts for length and monovalent salt but not for oligo concentration, terminal effects, denaturants (formamide, DMSO) or divalent cations such as Mg\u00b2\u207a, which significantly stabilise duplexes in PCR buffers.<\/li>\n<li><strong>For exact work, use the nearest-neighbour model:<\/strong> modern primer-design software uses nearest-neighbour thermodynamics, which considers the stacking energy of every dinucleotide step. The two formulas here are quick approximations accurate to a few degrees \u2014 ideal for planning and cross-checking, not for final assay design.<\/li>\n<li><strong>Interpreting the result:<\/strong> as a rule of thumb, hybridisation or annealing works best about 5 \u00b0C below the Tm. If the calculated Tm is far from your target, adjust the primer length or GC content rather than the annealing temperature alone.<\/li>\n<\/ul>\n<p>If you work with peptides or proteins in the same experiments, you may also find our <a href=\"https:\/\/wpcalc.com\/en\/chemistry\/isoelectric-point-calculator\/\">Isoelectric Point Calculator<\/a> useful for planning electrophoresis conditions, and the <a href=\"https:\/\/wpcalc.com\/en\/chemistry\/buffer-capacity-calculator\/\">Buffer Capacity Calculator<\/a> helps you check whether your hybridisation buffer holds its pH during heating.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Estimate the melting temperature (Tm) of a DNA oligonucleotide from its sequence and salt concentration. Enter one strand (A, C, G, T) and the Na\u207a concentration to get the Tm and GC content.<\/p>\n","protected":false},"author":3168,"featured_media":0,"comment_status":"open","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[54],"tags":[60],"class_list":["post-66885","post","type-post","status-publish","format-standard","hentry","category-chemistry","tag-biochemistry"],"acf":[],"_links":{"self":[{"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/posts\/66885","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/users\/3168"}],"replies":[{"embeddable":true,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/comments?post=66885"}],"version-history":[{"count":1,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/posts\/66885\/revisions"}],"predecessor-version":[{"id":66886,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/posts\/66885\/revisions\/66886"}],"wp:attachment":[{"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/media?parent=66885"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/categories?post=66885"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/wpcalc.com\/en\/wp-json\/wp\/v2\/tags?post=66885"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}