PCR Product Oligo Design Calculation

This form should be used to calculate oligonucleotide pairs meant for priming PCR reactions from template DNA (cDNA clone or genomic) sequence. The input should be coding sequence, and Osprey will attempt to generate primers specific to the target, similar in melting temperature, and producing reasonably sized products. When choosing a melting temperature (Tm) for your PCR reaction, remember that annealing is generally considered 5 degrees below Tm, under standard PCR conditions.

The thermodynamics used in these calculations are described here.

Target Sequence Data

This should be one DNA template sequence that is the target for PCR amplification.

Choose one of:
  1. Paste in DNA sequence (FastA/Pearson format preferred, other formats may parse with less confidence):
  2. Upload a target DNA sequence file (max 10MB):
Upload a secondary binding DNA sequence file, e.g. other transcripts from the same species (max 10MB):
Core Parameters
Perfect duplex melting temperature (Celsius)
Minimum Allowable difference for the pair Maximum
Oligonucleotide length
Minimum Optimal Maximum
Na+ concentration (molar) (standard PCR is typically 0.05M)
Other Parameters
PCR product length: Minimum Optimum Maximum
Product must include the following target position or interval
  • (e.g. 314 or 100-200, separate multiple products with commas):
  • or specified as a tab-delimited file:
  • or have a 5' or 3' bias
DNA concentration (molar)
Hairpin maximum (kcal/mol)
Dimer maximum (kcal/mol)
Secondary binding
Minimum margin between target melting temperature and closest secondary match (e.g. using default of 8, if target matches at 56°C, no secondary duplexes of >48°C)
Maximum free energy of binding (kcal/mol)
Minimum match to be considered a repeat region
Contiguous identical bases Overall percentage identity