Gene ruler ladder plus 100bp

In the CPE positive samples cell culture fluid was subjected to PCR and NPCR for confirmation of CPV. The DNA from the samples was extracted [11 ] and subjected to PCR by adding 15 µl of the template DNA, 5.0 µl of 10X PCR buffer (with 15 mM MgCl₂), 1.0 µl of forward and reverse primer (25 pm/µl) each, 1.0 µl of dNTPs mix (10 mM each), 0.5 µl of MgCl₂ (50mM), 1 U Taq DNA polymerase to make the final reaction of 50 µl using nuclease free water [12 ]. The reaction was put in a thermocycler (Veriti^®, Life Technologies, USA) with 35 cycles of denaturation at 94°C for 60s, annealing at 55°C for 60 s, elongation at 72°C for 150 s and a final elongation at 72°C for 10 min. For the NPCR, primers used were as per Mizak and Rzezutka [12 ] following the same conditions as of PCR. NPCR reaction was set up by adding 5 µl of the PCR product, 2.5 µl of 10X PCR buffer (with 15 mM MgCl₂), 1.0 µl each of forward and reverse primer (25 pm/µl), 1.0 µl of dNTPs (10 mM each), 0.5µl MgCl₂ (50 mM), 1 U Taq DNA polymerase and the final volume was made up to 25 µl by adding nuclease-free water.
PCR and NPCR products (10 µl) were run using 1% agarose with ethidium bromide at 5 volts/cm with Gene Ruler ladder plus 100bp (New England Biolabs, USA). The gel was visualized and photographed using Gel documentation system (AlphaImager, USA).

The primers for NPCR for the detection of CPV were as per Mizak and Rzezutka [10 ]. The NPCR reaction mixture was prepared by adding 5 μL of the PCR product (from above), 2.5 μL of 10× PCR buffer (with 15 mM MgCl₂), 1.0 μL each of forward and reverse primer (20 pm/μL), 1.0 μL of dNTPs (10 mM each) (Takara Bio), 0.2 μL Taq DNA polymerase (5 units/μL) Qiagen), and the final volume was made up to 25 μL by adding nuclease-free water.
Both PCR and NPCR were set at thermocycling parameters with 35 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 60 s, elongation at 72°C for 150 s, and a final elongation at 72°C for 10 min. Both PCR and NPCR products (10 μL each) were subjected to agarose gel electrophoresis using 1% agarose at the rate of 5 volts/cm with Gene Ruler ladder plus 100 bp (New England Biolabs, USA). The products on gel were visualized and documented using gel documentation system (Syngene, USA).