Nanodrop one onec uv vis spectrophotometer

The restriction endonuclease analysis (REA) was performed with 4 field isolates (KY748023, KY748022, KY748020, and KY748021). After virus inoculation in MDBK cells for 7–10 days and the CPEs had reached 90%, our viruses were collected followed by 3 freeze-thaw cycles and centrifugation at 5000×g for 15 min at 4 °C. The collected viruses were purified by centrifugation at 25,000×g for 5 h at 4 °C through 40% sucrose in 1× PBS before the pellets were resuspended in 550 μl of molecular grade water. To release the viral DNA, 35 μl of 20% SDS plus 40 μl of 20 mg/mL proteinase K were added to the virus suspension and incubated for 1 h at 37 °C. Then two rounds of phenol-chloroform extraction followed by ethanol precipitation were performed before the pellet was resuspended in TE buffer (pH 8.5) and an additional 0.2 μl of 10 μg/mL RNaseA and incubated for 30 min at 37 °C. After measurement by NanoDrop One/One^cUV-Vis Spectrophotometer (ThermoScientific, USA), the viral DNA was stored at + 4 °C. For REA, fastdigest Hind III, BamHI, and EcoRI (ThermoScientific, USA) restriction enzymes were used according to the manufacturer’s instructions. To visualize viruses, the cut viral DNAs were run in 0.6% agarose gel at 40 mV for 5–8 h with 1 μl of 10 mg/mL Ethidium Bromide per 20 mL of agarose gel.