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: ahya_s0044.g3.t1 (ahya_s0044.g3.t1), 4,485 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 4,485 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 AGGGCATTCGTGAAGTCGTT 4144–4163 20 59.965 50.000 AAAATGAAAACCACCCCGCG 4401–4420 20 59.967 50.000 277 0.068
2 CTCTCAAGGTGGCGGAACAT 1828–1847 20 60.036 55.000 CTTCAGATTGGAGCTGGGCA 2078–2097 20 60.035 55.000 270 0.071
3 CTCTCAAGGTGGCGGAACAT 1828–1847 20 60.036 55.000 TGGCTTCAGATTGGAGCTGG 2081–2100 20 60.035 55.000 273 0.071
4 TGTGCATGAGTTGAGCCGAT 830–849 20 60.036 50.000 TGGCAGCTTGATCATGAGCA 1100–1119 20 60.035 50.000 290 0.071
5 ACACGATCAGCGGCAGTTAA 4063–4082 20 60.038 50.000 AACGACTTCACGAATGCCCT 4144–4163 20 59.965 50.000 101 0.073
Primer map pair 1 (lowest penalty) · product 277 bp at template position 4,144–4,420 · the template track shows a 1,500 bp window of the 4,485 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 2,986–4,485 of 4,485Template1,500 bp windowproduct 277 bp · template 4,144–4,420product 277 bpleft primer, template 4,144–4,163right primer anneals here, template 4,401–4,4202,9863,3613,7364,1104,485Amplicon277 bpleft primer AGGGCATTCGTGAAGTCGTT · 5′→3′right primer AAAATGAAAACCACCCCGCG · its reverse complement binds the template strandinterior 237 bp4,1444,2134,2824,3514,420
Left primer
AGGGCATTCGTGAAGTCGTT
template 4,144–4,163 · 20 bp · Tm 59.965 °C · GC 50.000 %
Right primer
AAAATGAAAACCACCCCGCG
template 4,401–4,420 · 20 bp · Tm 59.967 °C · GC 50.000 %

Amplicon sequence (5′→3′ on the template strand, 277 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.

AGGGCATTCGTGAAGTCGTTGCATGTTCCAATTCATAAATAAATTTTAATGCATGTACAGAATGTATATATAGGCCTTTAAAGTGTTATTTTCTGGTTTATGATGAAACTGTCTGTGATTTGTATGTAAGTGAGACCGCAGAAAAATCGGATAGTTTGAAGCGCTTGTATTCATTGCCCGCATGCGCGGCTGATTGTCGCTCTTGACCACTATATTTGCTCTTCTTAATATCTTGTTTGTTTTCCCGTTGTTTCTCGCGCGGGGTGGTTTTCATTTT

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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