High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Goodman, Laura B.; Anderson, Renee R.; Slater, Marcia; Ortenberg, Elen; Renshaw, Randall W.; Chilson, Brittany D.; Laverack, Melissa A.; Beeby, John S.; Dubovi, Edward J.; Glaser, Amy L.

1. Once all of the above steps are completed put on new, well-fitted double gloves and sleeve covers. Then, add 2.5 µl of amplification master mix to a 384-well plate. 2. Transfer 2.5 µl of diluted preamp product to the 384-well plate according to the map in Table 2 and immediately cover the plate tightly with a foil seal. Discard outer gloves and sleeve covers. 3. Centrifuge the 384-well plate for 20 sec in the PCR plate spinner. 4. Do not remove the foil seal, but place the 384-well plate in the liquid handler. 5. Open the package containing an encased amplification plate and place it carefully in the liquid handler in the first slot with the serial number on the right -hold the case by the edges without touching the surface of the plate. Use unwrapped plates within one hour of opening. Note: The purpose of the case is to protect the plate from being touched, since this could disturb the small amounts of primers and probes inside the through-holes. 6. Remove the foil seal from the 384-well plate inside the liquid handler once everything is in place. Click Next and then check all the boxes as each step is completed. Click OK to start the run. 7. Close the door to the liquid handler and immediately start the filling process. Stay next to the liquid handler and immediately proceed to the next step once the plate is filled. 8. While the liquid handler is running, remove the protective film from the bottom of a case lid.