Table 1 Composition of PCR-RFLP reaction mix and PCR conditions
PCR reaction mix | ||
Taq buffer (10×) | 2.5 μL | |
MgCl2 (25 mM) | 2.5 μL | |
dNTP mix (20 mM) | 2.5 μL | |
Forward primer (10 μM) | 1.5 μL | |
Reverse primer (10 μM) | 1.5 μL | |
Taq DNA polymerase (5 units per μL) | 0.5 μL | |
Water | 12.0 μL | |
Genomic DNA | 2.0 μL | |
Total volume | 25.0 μL | |
PCR conditions | ||
Denaturation initial | 94 °C for 2 min | 01 cycle |
Denaturation subsequent cycles each | 94 °C for 30 s | 35 cycles |
Annealing | 66 °C for 30 s | |
Elongation initial | 72 °C for 30 s | |
Elongation final | 72 °C for 5 min | 01 cycle |
RFLP reaction mix | ||
PCR product | 10.0 μL | |
Nuclease-free water | 18.0 μL | |
Tango buffer 10× | 2.0 μL | |
BtsCI enzyme | 1.0 μL | |
Total volume | 31.0 μL | |