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: Actinostola_09424 (Actinostola_09424), 1,047 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 1,047 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 CCTTTCGGTGGGACGACTAC 784–803 20 60.109 60.000 CTGCATTCCAACCATCGCTG 902–921 20 59.900 55.000 138 0.209
2 AAGTTTTCTGTGGCAAGCGC 13–32 20 60.249 50.000 TGCGGGAAAACCTCAAACCT 181–200 20 60.106 50.000 188 0.355
3 CCTTTCGGTGGGACGACTAC 784–803 20 60.109 60.000 TCGACTCGGGGTGGTTTCTA 951–970 20 60.251 55.000 187 0.360
4 CCTTTCGGTGGGACGACTAC 784–803 20 60.109 60.000 TGATGGAGGTCTGGGATCGA 967–986 20 59.738 55.000 203 0.371
5 CCTTTCGGTGGGACGACTAC 784–803 20 60.109 60.000 ATGGAGGTCTGGGATCGACT 965–984 20 59.737 55.000 201 0.372
Primer map pair 1 (lowest penalty) · product 138 bp at template position 784–921 · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProduct

Template1,047 bpproduct 138 bp · template 784–921product 138 bpleft primer, template 784–803right primer anneals here, template 902–92112635247861,047Amplicon138 bpleft primer CCTTTCGGTGGGACGACTAC · 5′→3′right primer CTGCATTCCAACCATCGCTG · its reverse complement binds the template strandCCTTTCGGTGGGACGACTACCTGCATTCCAACCATCGCTGinterior 98 bp784818853887921
Left primer
CCTTTCGGTGGGACGACTAC
template 784–803 · 20 bp · Tm 60.109 °C · GC 60.000 %
Right primer
CTGCATTCCAACCATCGCTG
template 902–921 · 20 bp · Tm 59.900 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 138 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.

CCTTTCGGTGGGACGACTACTTATCGAGGTGCTAGATTAATGCCATCTATAGGATTAATGAATGGTGGTATACCGTTGTTCCATCACAGAGAACTTACTTTTATCAAACCAAGATTACCAGCGATGGTTGGAATGCAG

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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