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: Ahemp_017351-T1 (Ahemp_017351-T1), 5,108 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 5,108 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 CCAAGCACCTTGAAGCAACC 3262–3281 20 59.967 55.000 ATTGCTGAGGTTCCTCCAGC 3353–3372 20 60.035 55.000 111 0.067
2 ATCTTGCGCTGCTCAGTTCT 3709–3728 20 60.037 50.000 TTGTCGTCCAACTCCTCAGC 3843–3862 20 59.966 55.000 154 0.070
3 ATGCATGACCAAGGCGAAGA 4430–4449 20 60.035 50.000 GCCTCCTCTGGGTTTTCCTC 4542–4561 20 60.035 60.000 132 0.070
4 TGACAAAGCTCTCGGTGCAT 4394–4413 20 59.965 50.000 GCCTCCTCTGGGTTTTCCTC 4542–4561 20 60.035 60.000 168 0.070
5 ATGACAAAGCTCTCGGTGCA 4393–4412 20 59.965 50.000 GCCTCCTCTGGGTTTTCCTC 4542–4561 20 60.035 60.000 169 0.070
Primer map pair 1 (lowest penalty) · product 111 bp at template position 3,262–3,372 · the template track shows a 1,500 bp window of the 5,108 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 2,567–4,066 of 5,108Template1,500 bp windowproduct 111 bp · template 3,262–3,372product 111 bpleft primer, template 3,262–3,281right primer anneals here, template 3,353–3,3722,5672,9423,3173,6914,066Amplicon111 bpleft primer CCAAGCACCTTGAAGCAACC · 5′→3′right primer ATTGCTGAGGTTCCTCCAGC · its reverse complement binds the template strandCCAAGCACCTTGAAGCAACCATTGCTGAGGTTCCTCCAGCinterior 71 bp3,2623,2903,3173,3453,372
Left primer
CCAAGCACCTTGAAGCAACC
template 3,262–3,281 · 20 bp · Tm 59.967 °C · GC 55.000 %
Right primer
ATTGCTGAGGTTCCTCCAGC
template 3,353–3,372 · 20 bp · Tm 60.035 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 111 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.

CCAAGCACCTTGAAGCAACCGGGAACATTCCGGCGGCAATTGTCAATTTTGAGAAGGCAGATACTCATCGATTTGAAGTTCCTAGAATGTTGCTGGAGGAACCTCAGCAAT

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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