Inactivating porcine coronavirus before nuclei acid isolation with the temperature higher than 56 °C damages its genome integrity seriously

Qingxin Zhang; Qingshun Zhao

The mixture was vortexed thoroughly and then transferred into a column placed onto a 2 mL EP collection tube. After centrifuge for 1 min at 12,000×g, the column was placed onto a fresh 2 mL EP collection tube, followed by adding 600 µL washing buffer and then centrifuge for 30 sec at 12,000×g. Washing step was repeated once. The column was placed onto a new 2 mL collection. After centrifuge for 2 min at 12,000×g, the column was then placed onto a 1.5 ml collection tube. 100 µL and 40 µL elution buffer was added into the center of the membrane of column collected the sample prepared by R503 and Hank's solution respectively and then centrifuged for 1 min at 12,000 × g to collect the nucleic acid. The nucleic acid was stored at -30 ~ -15 o C for usage within short time and at -70 o C for long storage 2.3 Agarose gel electrophoresis 5 µL of the extracted nucleic acids, 1 µL of 10 × DNA loading buffer (P022, Vazyme) and 4 µL of nuclease-free H 2 O were mixed and then vortexed briefly. The mixture was heated in PCR instrument to denature the nucleic acid for 2 min and then subjected to 1.2% agarose gel electrophoresis separation on ice for 7-10 min together with the standard DNA marker (MD103, Vazyme).