CRITERIUM # DESCRIPTION OPTIMAL RANGE ACCEPTABLE RANGE
I length fragment < 100 bp <300bp
length primers 17-25 bp 17-30 bp
II Theoretical melt temperature >55°C - <60°C >50°C - <65°C
range of variation in melt temperature for developed toolkit < 5 °C 5 °C - 10 °C
III GC% 40-60 30-70
IV ΔG hairpin, self-dimer, hetero dimer < 6.00 < 9.00
bases suitable for binding with own primer sequence <6 <10
V BLAST identity >90% >99%
VI Mismatches both primer site closely related species (Ireland) >10 >6
Mismatches each primer site (within first 5 basepairs on 3’) >3 >1