Set 1 Set 2 Set 3 Set 4 Set 5
Reaction Mixture (reaction volume – 25µl )
Reaction Buffer (10X)a 1.0 1.0 1.0 1.0 1.0
MgCl2 (50mM)a 2.0 2.0 2.0 2.0 1.5
dNTP Mix (each) (10mM)a 0.2 0.2 0.2 0.2 0.2
DNA Polymerase (1U)a 1.5 1.0 1.0 1.0 1.0
Primers (each) (nM) Refer Table 3
First-PCR product (mL) 0.2 0.2 0.2 0.2 0.2
Cycle Condition          
Pre-denaturation (°C:min) 94:4 94:4 94:4 94:4 94:4
Denaturation (°C:sec) 94:30 94:30 94:30 94:30 94:30
Annealing (°C:sec) 60b:30 58c:30 64:30 58c:30 58:30
Extension (°C:sec) 72:30 72:30 72:30 72:30 72:30
Number of cycles 16 16 15 16 15
Final extension (°C:min) 72:7 72:7 72:7 72:7 72:7
Hold (°C:min) 20:∞ 20:∞ 20:∞ 20:∞ 20:∞
a   Biotools Company (Spain)
b   Touchdown from 60°C to 53.6°C (reduce by 0.4°C every cycle)
c   Touchdown from 58°C to 52°C (reduce by 0.4°C every cycle)
Table 2: Reaction mixtures and cycle condition for allele-specific second PCR.