Resources
A plain-language walkthrough of every setting in the primer & probe design tool and how to interpret what it returns. Each section below matches a panel in the tool, and the ? icons in the tool link directly to the relevant entry here.
Everything starts from the target sequence. Paste the region of the genome or transcript you want to amplify.
The DNA template you want to design primers against. Paste plain bases or FASTA text (a '>' header line followed by sequence). Spaces, digits and line breaks are ignored.
A short name for your target. It is used in the results, CSV download file name and the custom-assay enquiry text.
Choose which oligos the tool should design, or force it to use primers you already have.
When on, the tool searches for a forward primer on the plus strand.
When on, the tool also designs an internal hydrolysis (TaqMan-style) probe between the two primers.
When on, the tool searches for a reverse primer (reported 5'→3' as you would order it).
Force a specific forward primer sequence (5'→3'). The tool then only designs the probe and reverse primer around it.
Force a specific reverse primer sequence (5'→3').
High-level constraints applied before any thermodynamic checks.
Allowed amplicon lengths, as space-separated ranges such as '70-150 150-250'. The tool tries the first range first and only moves to the next when nothing fits.
How many ranked primer sets the tool reports back.
Maximum allowed stability (ΔG penalty score) of the primer's 3' end. A 3' end that binds too strongly misprimes easily.
How often a primer is allowed to match elsewhere in the template you pasted. Repeats cause the same primer to bind multiple places.
Size, melting temperature, GC content and secondary-structure limits applied to both primers.
Allowed primer length in bases. 'Opt' is the length the scorer prefers.
Allowed melting temperature range (°C), calculated with SantaLucia nearest-neighbour parameters and your salt conditions.
Largest allowed Tm gap between the left and right primer of a pair.
Allowed percentage of G and C bases in each primer.
Longest allowed run of a single identical base (e.g. AAAAA).
Limit for a primer binding to itself anywhere (self-dimer). Higher scores mean more stable self-dimers.
Limit for self-complementarity involving the primer's 3' end — the end the polymerase extends, so 3' dimers are extended into primer-dimer artefacts.
Limit for intramolecular hairpin (stem-loop) stability within one primer.
How many ambiguous (N) bases a primer may contain.
Number of G or C bases required within the last 5 bases at the 3' end. A GC clamp anchors the 3' end for efficient extension.
These values feed the Tm calculation — set them to match your actual master mix, not the defaults of another protocol.
Concentration of K⁺/Na⁺ in the reaction. Salt stabilises duplexes and raises Tm.
Mg²⁺ concentration. Mg²⁺ stabilises duplexes much more strongly than monovalent salt.
dNTP concentration. dNTPs chelate Mg²⁺, reducing its effective concentration.
Concentration of each primer in the reaction; used in the Tm calculation.
The annealing temperature you plan to run. Used as a reference when scoring how far each oligo's Tm sits from your cycling conditions.
Constraints for the dual-labelled probe sitting between the two primers.
Allowed probe length in bases.
Allowed probe melting temperature. The probe must hybridise before the primers extend, so it needs a higher Tm than the primers.
Allowed GC content of the probe.
Longest single-base run allowed in the probe.
Self-dimer and 3' self-dimer limits for the probe.
Minimum Tm advantage the probe must have over the primers.
A 5' guanine sits directly next to the reporter dye and quenches its fluorescence even after cleavage.
Position constraints, written as 1-based start,length pairs exactly like Primer3web 0.4.0.
Restricts the search to a slice of the pasted sequence. Everything outside is ignored.
Regions the amplicon must overlap — e.g. a mutation site or a diagnostic signature. Space-separated start,length pairs.
Regions where no primer or probe may land — e.g. known SNPs or homologous stretches.
Individual 1-based positions that the amplicon must cover, comma- or space-separated.
Each returned set is a left primer, probe and right primer ranked by total penalty — lower is better.
A weighted sum of how far each oligo deviates from the optimum size, Tm and GC, plus structure penalties. The best set has the lowest penalty.
Melting temperature of the oligo under your salt and concentration settings.
Percentage of G+C in the oligo.
Self-dimer stability scores, anywhere in the oligo and at the critical 3' end.
Stability of the strongest stem-loop the oligo can fold into.
How strongly the two primers bind each other (primer-dimer risk), shown as 'pair complementarity' and '3' pair complementarity' under the table.
Amplicon length in bp and its 1-based start on your template.
How many candidate oligos were examined and why each was rejected (Tm, GC, poly-X, self-complementarity, hairpin, excluded regions…).
Start from set 1 (lowest penalty). Confirm the amplicon covers your target region, check the probe has no 5' G, and sanity-check specificity against the organism's genome (e.g. BLAST) before ordering.
Open the tool, paste your target sequence and click “Pick primers”. The design runs entirely in your browser — nothing is uploaded.