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PrimerCalc Primer Tm

Nearest-neighbor Tm · salt/conc. correction · dimer & hairpin checks

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Paste primer sequence(s)

One primer per line; "name sequence" and FASTA (>) both work; case-insensitive, U counts as T, spaces and line numbers are ignored.

Reaction conditions

Tm uses nearest-neighbor salt correction (0.368·N·ln[Na+]) and the R·ln(Ct/4) concentration term; typical PCR buffer has ~50 mM Na+, qPCR primers run at 200-500 nM.

Tm & structure overview

Parameter details

SantaLucia 1998 nearest-neighbor parameters

Stack values from SantaLucia, J. (1998) PNAS 95:1460-1465, Table 2 (unified parameters); initiation: +2.3 kcal/mol / +4.1 cal/(mol K) per terminal A-T pair, +0.1 / 0 per terminal G-C pair, plus a symmetry correction of -1.4 cal/(mol K) for non-self-complementary duplexes. Tm = dH*1000 / (dS' + R*ln(Ct/x)) - 273.15, with Ct = 2C and x = 4 for ordinary duplexes, Ct = C and x = 1 for self-complementary ones; dS' = dS + 0.368*N*ln[Na+].

What this tool is

PrimerCalc is an online Tm calculator for PCR primers and oligonucleotides. The headline number uses the nearest-neighbor thermodynamic model: SantaLucia 1998 unified stack parameters summed per dinucleotide, with terminal initiation and symmetry corrections, a Na+ entropy correction and the R*ln(Ct/x) concentration term. It also reports GC%, GC clamp, Wallace/basic-rule references, 3'-end self-complementarity, hairpins, homopolymers and design-rule warnings - essentially the core readouts of Thermo OligoAnalyzer and IDT OligoCalc, with batch support.

Everything runs in your own browser: the parameter tables ship with the page, there is no backend and no sequence ever leaves the tab. Your input is kept in local storage, and hundreds of primers still compute in milliseconds. Hairpin and dimer dG values are stack-only sums (no initiation term), the same local-stability convention MARMIA uses, so candidates compare fairly.

Features

How to use it

  1. Paste candidate primers, one per line; prefix a name like "F1 ATGG..." or use FASTA headers.
  2. Match your chemistry: typical Taq buffer ~50 mM Na+, qPCR primers 200-500 nM.
  3. Read the overview table: forward/reverse Tm differing by >5 C, 3' self-complementarity >=4 bp or hairpin stems >=4 bp all deserve a redesign.
  4. Check dH/dS, hairpin dG and homopolymer positions in the details table, then copy or download the results.

FAQ

Nearest-neighbor vs the Wallace rule - which should I trust?
The Wallace rule (2(A+T)+4(G+C)) only counts composition and treats every G-C pair identically; even under 14 nt it carries roughly +/-5 C of error. Nearest-neighbor sums per-stack thermodynamics with terminal, salt and concentration terms, typically landing within about 2 C of measured melting. Design and report with nearest-neighbor; keep Wallace as a sanity check.
Why does salt correction modify the entropy?
Cations screen the phosphate-phosphate repulsion that destabilizes the duplex. SantaLucia's treatment adds 0.368*N*ln[Na+] to dS (N = length), which then flows through the Tm denominator - closer to thermodynamics and to measurement than the old additive 16.6*log10[Na+] nudge on Tm. This tool uses the entropy convention.
How do I read the dimer and hairpin numbers?
3'-end self-complementarity means a suffix ending at the 3' OH can fully pair with another window (two primer molecules pairing is enough to seed a dimer): >=4 bp is a warning sign, >=8 bp almost guarantees one. A hairpin is the strand folding on itself; stems >=4 bp with 3-8 nt loops measurably slow polymerase extension. Both report a 25 C stack-only dG - more negative is more stable.
Why were N, I, X stripped from my sequence?
The NN model is defined for ACGT stacks only; degeneracy codes (N/R/Y...) and stray letters have no stack parameters, so they are removed and counted in the warning line instead of silently skewing the result. For degenerate primers, expand each variant and compute them separately.
Are my sequences uploaded?
No. Fully static page, no backend; sequences and parameters are computed locally and only your input is kept in this browser's localStorage. Unpublished primer sets are safe.

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