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: scaffold4.g62.t2 (scaffold4.g62.t2), 2,847 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 2,847 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 TAGATGATGTGGATGCCGCC 667–686 20 59.965 55.000 GGTGTTCGCCCAAACTTGTC 927–946 20 59.970 55.000 280 0.066
2 TCTGTTGGGTTACTTCGCCC 725–744 20 59.964 55.000 GGTGTTCGCCCAAACTTGTC 927–946 20 59.970 55.000 222 0.066
3 CGTCGCTCTGTTGGGTTACT 719–738 20 60.039 55.000 GGTGTTCGCCCAAACTTGTC 927–946 20 59.970 55.000 228 0.069
4 TAGATGATGTGGATGCCGCC 667–686 20 59.965 55.000 TCCCTTACAGCAGCGTGAAG 849–868 20 60.037 55.000 202 0.073
5 TAGATGATGTGGATGCCGCC 667–686 20 59.965 55.000 AAATGCATTGGTGTTCGCCC 936–955 20 60.037 50.000 289 0.073
Primer map pair 1 (lowest penalty) · product 280 bp at template position 667–946 · the template track shows a 1,500 bp window of the 2,847 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 57–1,556 of 2,847Template1,500 bp windowproduct 280 bp · template 667–946product 280 bpleft primer, template 667–686right primer anneals here, template 927–946574328071,1811,556Amplicon280 bpleft primer TAGATGATGTGGATGCCGCC · 5′→3′right primer GGTGTTCGCCCAAACTTGTC · its reverse complement binds the template strandinterior 240 bp667737807876946
Left primer
TAGATGATGTGGATGCCGCC
template 667–686 · 20 bp · Tm 59.965 °C · GC 55.000 %
Right primer
GGTGTTCGCCCAAACTTGTC
template 927–946 · 20 bp · Tm 59.970 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 280 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.

TAGATGATGTGGATGCCGCCATGGACATTACACACTTTGGAGCAGCAGGAAACGTCGCTCTGTTGGGTTACTTCGCCCGGCTTGCCACTAGTACTGAGAACGAGCTTGATATTCTGTACTTAGATTCCCTTAAAAGAGCGGGTGCTAACTATGATTTTCCGGATTTATATGGGCAGACGGCACTTCACGCTGCTGTAAGGGACTGGCATCCGGACGTGGCCAAGTATCTTCTTGATGAAGGCGCAAATGCTGCCGTTTGTGACAAGTTTGGGCGAACACC

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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