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: ANN26669-RA (ANN26669-RA), 2,541 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 2,541 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 TTTGAAAGAGCACGTGCACG 2142–2161 20 59.973 50.000 CGCATGGCATTCTGGTAACG 2386–2405 20 59.972 55.000 264 0.055
2 CGTTACCAGAATGCCATGCG 2386–2405 20 59.972 55.000 GGTGAAGATCCTCGTAGCCG 2485–2504 20 59.969 60.000 119 0.059
3 CGTTACCAGAATGCCATGCG 2386–2405 20 59.972 55.000 GCTGACTGCTGGGTGAAGAT 2496–2515 20 60.036 55.000 130 0.064
4 TACCACGCACTCCTAAGGGA 1121–1140 20 59.960 55.000 GGAGATATTCCCGTGCGAGG 1215–1234 20 60.038 60.000 114 0.078
5 TTTGAAAGAGCACGTGCACG 2142–2161 20 59.973 50.000 TGGTTTTGCTTTGGCGACAG 2297–2316 20 59.898 50.000 175 0.130
Primer map pair 1 (lowest penalty) · product 264 bp at template position 2,142–2,405 · the template track shows a 1,500 bp window of the 2,541 bp template — the blue segment on the top bar is where that window sits, and every scale is in template coordinates · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProductWindow drawn in the template track

whole templatethe 1,500 bp drawn in the track below · template 1,042–2,541 of 2,541Template1,500 bp windowproduct 264 bp · template 2,142–2,405product 264 bpleft primer, template 2,142–2,161right primer anneals here, template 2,386–2,4051,0421,4171,7922,1662,541Amplicon264 bpleft primer TTTGAAAGAGCACGTGCACG · 5′→3′right primer CGCATGGCATTCTGGTAACG · its reverse complement binds the template strandinterior 224 bp2,1422,2082,2742,3392,405
Left primer
TTTGAAAGAGCACGTGCACG
template 2,142–2,161 · 20 bp · Tm 59.973 °C · GC 50.000 %
Right primer
CGCATGGCATTCTGGTAACG
template 2,386–2,405 · 20 bp · Tm 59.972 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 264 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.

TTTGAAAGAGCACGTGCACGCACCTCCTTTTCAACCAATCACGTTTTCGATCACCAAGCAGTTGTCAATCAGATGCTTGTCACAGAATAAAACTGTCCTCACGTTCAATCAAGGAAAATACACAGCTCGTTTCAACGTCGGATCCAAGCTTAAGGCTGTCGCCAAAGCAAAACCACCAACAAGTAGAGACAGTTACGAGACACATCTTGCAGAAGTGAAGCAGTTTGTGAATATTGTCTTAGATCGTTACCAGAATGCCATGCG

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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