Sequences
Forward (5′→3′)
ATGACGAGGACAACGGTAGATReverse (5′→3′)
TCATACTTGGAGATCATCACGGTAmplicon
185 bp
Primer Tm (°C)
| Conditions | Forward | Reverse | Average | Δ F/R |
|---|---|---|---|---|
| qPCR master mix | 62.0 | 62.2 | 62.1 | 0.1 |
| Primer-BLAST default | 58.6 | 59.0 | 58.8 | 0.4 |
qPCR master mix: Owczarzy 2004/2008 · 250 nM primer · 50 mM Na⁺ · 3 mM Mg²⁺ · 0.8 mM dNTP
Primer-BLAST default: SantaLucia 1998 salt corr. · 50 nM primer · 50 mM Na⁺ · 1.5 mM Mg²⁺ · 0.6 mM dNTP
Bold, underlined base = 3′ end (polymerase extension point).
Primer QC
GC% · Forward
47.6%GC% · Reverse
43.5%Self-dimer ΔG · Fwd (any position)
-0.2 kcal/molSelf-dimer ΔG · Fwd (3′ end)
1.2 kcal/molSelf-dimer ΔG · Rev (any position)
-1.6 kcal/molSelf-dimer ΔG · Rev (3′ end)
0.5 kcal/molHeterodimer ΔG (F×R, any position)
-1.6 kcal/molHeterodimer ΔG (F×R, 3′ end)
-0.2 kcal/molΔG at 37 °C · SantaLucia 1998 NN parameters · < −6 kcal/mol = significant dimer risk · "3′ end" is restricted to duplexes anchored at a primer's own 3′ terminal base (polymerase-extendable); "any position" is the most stable duplex anywhere in the primer. Estimated screening value. For comprehensive dimer analysis, validate with Primer3.
Source
Citation
Xu et al. 2022Genomic mapping
Each row is one transcript, aligned on a shared axis. A gap means that isoform skips this exonic region. Each row's box is drawn at that isoform's own real exon length, which can be narrower than the column (alternative splice site); hover a box for its exact size. A ~amber header bp means isoforms don't all agree on this region's boundary, while a plain header means they do. Navy blocks mark where the primers bind, and green (with a ✓ by the name) means this isoform has a confirmed working amplicon, not just exon overlap. A row with a “‹ exons” / “exons ›” hint instead of any box means that isoform's exons exist but sit outside the current scroll position (a truncated isoform or alternate transcription start site) — click the hint to jump to them. Click a row to load it below.
★ = Ensembl canonical transcript (reference model, not necessarily the most expressed isoform)
Showing: ENSMUST00000233392
Off-target specificity
SpecificNo paired forward+reverse off-target amplicon found against nt (whole genome/transcriptome). Screen requires >=2 total mismatches including >=2 within the last 5bp at the 3' end to clear a hit (NCBI Primer-BLAST-style stringency); hits with >=6 total mismatches are dropped as implausible.
Efficiency measurements
Ideal range:Dilution series 1.90–2.10·LinRegPCR 1.70–1.90·unknown method: not scoredAnalyse your qPCR run with LinRegPCR efficiency correction and a MIQE 2.0 report.