8.3.1. Qualitative PCR
Procedure (after McKee et al., 2003):
- Genomic DNA (5-30 ng) is amplified using a
thermal cycler in a 50 µl reaction comprising:
- 4 mM MgCl2 ,
- 200 µM of each deoxyribonucleotide triphosphate,
- 100 ng of primers MP1 and MP2 (Table 2),
- 5 µl of 10 x PCR buffer (100 mM tris-HCl, pH 8.3; 500 mM KCL),
- 2-5 µg of Taq DNA polymerase.
- Conditions for amplification consist of:
- initial denaturation at 95o C for 2 min,
- 40 cycles of denaturation (95o C, 30 s),
- primer annealing (61o C, 15 s),
- primer extension (72o C, 60 s),
- final extension cycle (72o C, 5 min).
- Amplification products are analysed by electrophoresis (55 V, 1.5 h) through 1.0-1.5 % (wt / vol) agarose containing ethidium bromide. A 486 bp PCR product is produced from primers MP1 and MP2. To ensure test specificity; a second PCR following the same protocol (using primers MP1 and MP3) is conducted, and a specific 276 bp hemi-nested product is amplified from the 486 bp template.