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: amic_s0035.g10.t1 (amic_s0035.g10.t1), 1,637 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 1,637 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 CGGTGGGGGAGATCAAAACA 883–902 20 59.962 55.000 AGCTGACGTTTGGAGCGTTA 966–985 20 59.967 50.000 103 0.071
2 ATTCTTGCGCCAGTTCTGGA 1312–1331 20 59.963 50.000 TGTTTGGCACTTGTTCGCAG 1445–1464 20 59.900 50.000 153 0.137
3 ATTCTTGCGCCAGTTCTGGA 1312–1331 20 59.963 50.000 CTGTTTGGCACTTGTTCGCA 1446–1465 20 59.900 50.000 154 0.137
4 ATTCTTGCGCCAGTTCTGGA 1312–1331 20 59.963 50.000 CTCCTGTGATGATGCGTGGT 1569–1588 20 60.108 55.000 277 0.145
5 CGGTGGGGGAGATCAAAACA 883–902 20 59.962 55.000 GAAAGCTGACGTTTGGAGCG 969–988 20 60.111 55.000 106 0.148
Primer map pair 1 (lowest penalty) · product 103 bp at template position 883–985 · the template track shows a 1,500 bp window of the 1,637 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 138–1,637 of 1,637Template1,500 bp windowproduct 103 bp · template 883–985left primer, template 883–902right primer anneals here, template 966–9851385138881,2621,637Amplicon103 bpleft primer CGGTGGGGGAGATCAAAACA · 5′→3′right primer AGCTGACGTTTGGAGCGTTA · its reverse complement binds the template strandCGGTGGGGGAGATCAAAACAAGCTGACGTTTGGAGCGTTAinterior 63 bp883909934960985
Left primer
CGGTGGGGGAGATCAAAACA
template 883–902 · 20 bp · Tm 59.962 °C · GC 55.000 %
Right primer
AGCTGACGTTTGGAGCGTTA
template 966–985 · 20 bp · Tm 59.967 °C · GC 50.000 %

Amplicon sequence (5′→3′ on the template strand, 103 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.

CGGTGGGGGAGATCAAAACAAAAGTTTGGTTTTATCAAACGAGTTGATAAATGTTGAATTAGTCATTCGCTCTGACGAAGGGCTAACGCTCCAAACGTCAGCT

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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