Primer Design

Primers can be designed directly against the sequences annotated in CnidoSite, or against a sequence you paste in. Design is performed by primer3 (version 2.6.1) running on this server — your sequence is not sent to any third-party website. For reference, the primers released with this database were designed with an optimal melting temperature of 60°C (58–62 °C), an optimal primer length of 20 bp (18–24 bp), and a GC content between 40% and 60%, with self-complementarity restricted to limit primer-dimer formation; that parameter set is the CnidoSite published protocol preset below.

Template taken from CnidoSite: FUN_031411-T1 (FUN_031411-T1), 714 bp.
Template

Two ways to supply a template: pick a species and a gene / transcript ID, or paste your own sequence. If the gene ID is found it is used; the pasted sequence is used otherwise. A template must be at least 30 bp.

Load example sequence
Primer parameters
Advanced parameters (any field left empty keeps the preset value)

A value that is not a number is ignored. The product size range is applied only when both of its fields are filled in and the maximum is at least the minimum.

Melting temperature
Primer length and GC content
Product and output
Self-complementarity and probe
Download TSV
Design results sorted by primer3 penalty, lowest first

Template 714 bp · parameter preset CnidoSite published protocol (Tm 60 [58-62] °C, 20 [18-24] bp, GC 40-60%) · 5 primer pairs returned.

Pair Left primer (5'→3') Position Len Tm (°C) GC (%) Right primer (5'→3') Position Len Tm (°C) GC (%) Product (bp) Penalty
1 GAAGACGACGCGATTTCAGC 43–62 20 59.976 55.000 TGGGACTTGATTTCACGCCA 247–266 20 59.890 50.000 224 0.133
2 GCAACATGGTTGCTGCTTGA 112–131 20 59.968 50.000 TGGGACTTGATTTCACGCCA 247–266 20 59.890 50.000 155 0.142
3 CACAGCGCGATTTGTTGACA 473–492 20 60.041 50.000 TCCCTTCTTGAGTGGTCCCA 572–591 20 60.105 55.000 119 0.146
4 CACAGCGCGATTTGTTGACA 473–492 20 60.041 50.000 TGAGTGGTCCCAAAGTCGTG 564–583 20 59.894 55.000 111 0.147
5 CACAGCGCGATTTGTTGACA 473–492 20 60.041 50.000 GAGTGGTCCCAAAGTCGTGT 563–582 20 59.894 55.000 110 0.148
Primer map pair 1 (lowest penalty) · product 224 bp at template position 43–266 · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProduct

Template714 bpproduct 224 bp · template 43–266product 224 bpleft primer, template 43–62right primer anneals here, template 247–2661179358536714Amplicon224 bpleft primer GAAGACGACGCGATTTCAGC · 5′→3′right primer TGGGACTTGATTTCACGCCA · its reverse complement binds the template strandinterior 184 bp4399155210266
Left primer
GAAGACGACGCGATTTCAGC
template 43–62 · 20 bp · Tm 59.976 °C · GC 55.000 %
Right primer
TGGGACTTGATTTCACGCCA
template 247–266 · 20 bp · Tm 59.890 °C · GC 50.000 %

Amplicon sequence (5′→3′ on the template strand, 224 bp) — blue is the left primer, red is where the right primer anneals. Copy from here to order or to check a base by eye.

GAAGACGACGCGATTTCAGCACCCAGTTTTGTTCCTAAACTGCAAGCCTTCATAAACGGCCAACCCAAAGCAACATGGTTGCTGCTTGATGTAACAGTTCAGGGAAGCAAAGCCAAAGTTTATCACAGTCGAGATCTCAGCAACATTGCGTCTTATTTTTACCTAATGTACGAAGAAATGCCAATCGACTGGTTGATGGATTATTGGCGTGAAATCAAGTCCCA

The right primer is listed as its own 5'→3' sequence; what anneals to the template strand shown here is its reverse complement, so the highlighted stretch is complementary to the listed sequence. That is expected.

How to read this table. Position is a 1-based interval on the template, and both primers use the same convention: it is the stretch the primer occupies on the template strand (for the left primer, starting at its 5’ end; for the right primer, the region it anneals to, i.e. where the reverse complement of the listed sequence sits on the template). The two coordinates in a row can therefore be subtracted directly, and the amplicon view above marks both of them so you can check them by eye. Penalty is primer3’s weighted sum of the deviations from your constraints — lower is better, and the pairs are returned in that order. Tm is computed by primer3 from the SantaLucia thermodynamic parameters at the salt and primer concentrations in effect. The primer sequences themselves are 5’→3′ and can be ordered as they stand; the right primer anneals to the template as its reverse complement.

Specificity has not been assessed. This page only designs primers; it does not check whether they also anneal elsewhere in the genome or transcriptome. Once you have candidates, confirm each primer sequence with this site’s BLAST. The Tm and GC settings above are the design targets, and the annealing temperature still has to be optimised at the bench.

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