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Design Guidelines

Target properties

  • 100-200bp with SYBR Green.
  • 60-120bp with all probe-based methods.
  • With cDNA templates, target sequence should span exon-exon junctions to avoid amplifying any contaminating genomic DNA.

Primer properties

  • Length: 18-30bp.
  • % GC: 20-80%.
  • Both primers should have the same Tm (55oC - 60oC).
  • For TaqMan® experiments, Tm of primers should be above 60oC.
  • No 3' GC clamps
  • When using software to design primers, select 100mM as the monovalent cation concentration and 5mM as the Mg++ concentration.
  • It is recommended that primers sequences be checked with BLAST to make sure they do not hybridize to unintended targets.

Probe properties

  • Length: 20-30bp.
  • % GC: 20-80% (more C's than G's).
  • The Tm should be 10oC above the primers'.
  • No 3 base runs especially G's.
  • No 5' end G.
  • 5' end of probe should be within 3 bp of 3' end of primer on same strand (10-12bp max.).
  • Match dye and quencher properly. Black Hole Quenchers are recommended.
  • Additional factors for Molecular Beacons®
    • Start with design of standard hydrolysis probe (i.e. TaqMan®).
    • To chosen sequence add 5-7bp stem with Tm similar to probe.
    • Test folding with Mfold.
    • Avoid complimentarity between primers and probe.
    • It is recommended that Tm of primers and probe be verified experimentally.

Dye parameters

  • Avoid spectral overlap (i.e. FAM + TET or TET + VIC are bad choices).
  • If using four dyes FAM - HEX - ROX - CY5 is a good combination.

Primer & Probe Parameters

  • For sequence specific reactions (i.e. TaqMan®), desalted primers are adequate.
  • For SYBR green reactions, HPLC purified primers are recommended.
  • Primer stock solutions should be between 6-18µM.
  • Probe oligos should be HPLC purified.
  • Working stock aliquots should be between 2-6µM (or 20X reaction concentration of 100-300nM for each primer).