- 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.
- 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.
- 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
- Additional factors for Molecular Beacons®
- Start with design of standard hydrolysis probe (i.e.
- To chosen sequence add 5-7bp stem with Tm similar to
- Test folding with Mfold.
- Avoid complimentarity between primers and probe.
- It is recommended that Tm of primers and probe be
- Avoid spectral overlap (i.e. FAM + TET or TET + VIC are
- If using four dyes FAM - HEX - ROX - CY5 is a good
Primer & Probe Parameters
- For sequence specific reactions (i.e. TaqMan®), desalted
primers are adequate.
- For SYBR green reactions, HPLC purified primers are
- 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).