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: ANN22216-RA (ANN22216-RA), 2,844 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 2,844 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 CGCGGAATCGACCAGATACA 1927–1946 20 59.970 55.000 CGAGGTATTTTTCTGGCGCG 2103–2122 20 59.974 55.000 196 0.057
2 CGCGCCAGAAAAATACCTCG 2103–2122 20 59.974 55.000 TAATACGGATCCTGCGTGCC 2299–2318 20 59.967 55.000 216 0.059
3 CGGAAGGTTCACGTCCTGAA 1823–1842 20 59.967 55.000 CGAGGTATTTTTCTGGCGCG 2103–2122 20 59.974 55.000 300 0.059
4 CGCGCCAGAAAAATACCTCG 2103–2122 20 59.974 55.000 TTCAGTTTCCCAGCGGTACC 2323–2342 20 59.964 55.000 240 0.062
5 CGCGCCAGAAAAATACCTCG 2103–2122 20 59.974 55.000 TGCGTGCCATCTAACCAGTT 2287–2306 20 59.964 50.000 204 0.062
Primer map pair 1 (lowest penalty) · product 196 bp at template position 1,927–2,122 · the template track shows a 1,500 bp window of the 2,844 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 1,275–2,774 of 2,844Template1,500 bp windowproduct 196 bp · template 1,927–2,122product 196 bpleft primer, template 1,927–1,946right primer anneals here, template 2,103–2,1221,2751,6502,0252,3992,774Amplicon196 bpleft primer CGCGGAATCGACCAGATACA · 5′→3′right primer CGAGGTATTTTTCTGGCGCG · its reverse complement binds the template strandinterior 156 bp1,9271,9762,0252,0732,122
Left primer
CGCGGAATCGACCAGATACA
template 1,927–1,946 · 20 bp · Tm 59.970 °C · GC 55.000 %
Right primer
CGAGGTATTTTTCTGGCGCG
template 2,103–2,122 · 20 bp · Tm 59.974 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 196 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.

CGCGGAATCGACCAGATACACGATCTCTGGGAAAACGGCGAAGAGATTTACCGGAACGGTGAATACTTCACCGAACTCATCACAGCATATGCCATCAGATATATCCGTAAGTCTGTTGAACTCGGCAAGCCTTTTTTTATCTACGTACCTTACAACGCGCCACATTACCCGATGCACGCGCCAGAAAAATACCTCG

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.

TOP