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: aflo_s0017.g84.t1 (aflo_s0017.g84.t1), 1,685 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 1,685 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 TTGGACGTTCATCCTCGCAA 1076–1095 20 59.966 50.000 GAGCTGCGGCAGAATCAATG 1174–1193 20 59.971 55.000 118 0.063
2 ACTCCTCCTGATGCAGTTGC 1043–1062 20 60.036 55.000 GAGCTGCGGCAGAATCAATG 1174–1193 20 59.971 55.000 151 0.065
3 CATTGATTCTGCCGCAGCTC 1174–1193 20 59.971 55.000 ATCAACGGGGTGTAGCGTTT 1442–1461 20 59.964 50.000 288 0.065
4 GGGGTGGCTCAGATTGTCAA 311–330 20 59.962 55.000 GATCACTGCAAGAGCGCATG 453–472 20 59.972 55.000 162 0.066
5 CATTGATTCTGCCGCAGCTC 1174–1193 20 59.971 55.000 GTCAAGAACCACGCTCGGTA 1317–1336 20 60.039 55.000 163 0.068
Primer map pair 1 (lowest penalty) · product 118 bp at template position 1,076–1,193 · the template track shows a 1,500 bp window of the 1,685 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 186–1,685 of 1,685Template1,500 bp windowproduct 118 bp · template 1,076–1,193product 118 bpleft primer, template 1,076–1,095right primer anneals here, template 1,174–1,1931865619361,3101,685Amplicon118 bpleft primer TTGGACGTTCATCCTCGCAA · 5′→3′right primer GAGCTGCGGCAGAATCAATG · its reverse complement binds the template strandTTGGACGTTCATCCTCGCAAGAGCTGCGGCAGAATCAATGinterior 78 bp1,0761,1051,1351,1641,193
Left primer
TTGGACGTTCATCCTCGCAA
template 1,076–1,095 · 20 bp · Tm 59.966 °C · GC 50.000 %
Right primer
GAGCTGCGGCAGAATCAATG
template 1,174–1,193 · 20 bp · Tm 59.971 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 118 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.

TTGGACGTTCATCCTCGCAATTTCAGCGATTTTGAATTCATGCTTCGTGTAAACTTACACGATTTGGTGCACTGCTTGATCGATGGTACCATGTGTACCATTGATTCTGCCGCAGCTC

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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