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: OS493_026545-T1 (OS493_026545-T1), 231 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 231 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 TGGTTTGGGTGCAATCTCCA 63–82 20 59.813 50.000 TGTCACTTGACTCCTTTCGGT 173–193 21 59.239 47.619 131 1.948
2 TGGTTTGGGTGCAATCTCCA 63–82 20 59.813 50.000 TCACTTGACTCCTTTCGGTCA 171–191 21 58.964 47.619 129 2.224
3 TGGTTTGGGTGCAATCTCCA 63–82 20 59.813 50.000 ACTTGACTCCTTTCGGTCACT 169–189 21 58.960 47.619 127 2.227
4 TGGTTTGGGTGCAATCTCCA 63–82 20 59.813 50.000 CACTTGACTCCTTTCGGTCAC 170–190 21 58.864 52.381 128 2.323
5 TGGTTTGGGTGCAATCTCCA 63–82 20 59.813 50.000 TTGTCACTTGACTCCTTTCGGT 173–194 22 59.830 45.455 132 2.357
Primer map pair 1 (lowest penalty) · product 131 bp at template position 63–193 · 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

Template231 bpproduct 131 bp · template 63–193product 131 bpleft primer, template 63–82right primer anneals here, template 173–193159116174231Amplicon131 bpleft primer TGGTTTGGGTGCAATCTCCA · 5′→3′right primer TGTCACTTGACTCCTTTCGGT · its reverse complement binds the template strandTGGTTTGGGTGCAATCTCCATGTCACTTGACTCCTTTCGGTinterior 90 bp6396128161193
Left primer
TGGTTTGGGTGCAATCTCCA
template 63–82 · 20 bp · Tm 59.813 °C · GC 50.000 %
Right primer
TGTCACTTGACTCCTTTCGGT
template 173–193 · 21 bp · Tm 59.239 °C · GC 47.619 %

Amplicon sequence (5′→3′ on the template strand, 131 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.

TGGTTTGGGTGCAATCTCCATTCTATTCGACATGTTATTTATTGTTCAACACTATTGCCTTTATGGATCAAAGCACGCCAGATATGAAGTGATCATTGATGACAGTAGTGACCGAAAGGAGTCAAGTGACA

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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