Resources

Primer & probe design

The familiar Primer3web 0.4.0 inputs, in our styling, tuned for hydrolysis-probe qPCR. Everything runs inside your browser — the sequence is never uploaded, and results are calculated on your own machine.

Which engine you are running: this is the JavaScript engine written to Primer3's rules — SantaLucia nearest-neighbour Tm with monovalent/divalent salt correction, complementarity, hairpin and 3' stability scanning, and penalty-weight ranking. It is not the compiled Primer3 binary, so penalty values can differ slightly from primer3.org output. For research use only.

Full guide: what every parameter does and how to read the results — or click any ? icon next to a setting.

Template sequence

Sequence ID

0 bases read. Plain sequence or FASTA; spaces, digits and line breaks are ignored.

What to pick

Pick left primerPick hybridisation probePick right primer

Or use this left primerOr use this right primer

Product & general settings

Product size rangesNumber to returnMax 3' stabilityMax mispriming (in template)Primer size minPrimer size optPrimer size maxPrimer Tm minPrimer Tm optPrimer Tm maxMax Tm differenceMin GC %Opt GC %Max GC %Max poly-XMax self complementarityMax 3' self complementarityMax hairpinMax #NGC clamp

Reaction conditions

Monovalent salt (mM)Divalent salt (mM)dNTPs (mM)Primer concentration (nM)Annealing temperature (°C)

Internal oligo (hydrolysis probe)

Probe size minProbe size optProbe size maxProbe Tm minProbe Tm optProbe Tm maxProbe min GC %Probe max GC %Probe max poly-XProbe max self compl.Probe max 3' self compl.Probe Tm above primers (°C)

Reject probes with G at the 5' end

Regions

Included region (start,length)Targets (start,length …)Excluded regions (start,length …)Must include these positions

Positions are 1-based, exactly as in Primer3web 0.4.0.