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. Remove the appropriate number of amplification plates from the freezer (up to 4 may be run at once). Do not open the packaging. Allow the plate to warm up for at least 15 min at room temperature. Record the serial number and lot number of the plate. NOTE: Unopened plates can remain at room temperature for up to 24 hr, but for best practice, remove these from the freezer no more than 30 min before needed. 2. Centrifuge the completed preamp plate for 20 sec using the PCR plate spinner. Turn on the amplification machine and computer and start the associated program. 3. Remove all unnecessary items from a PCR setup area. Put on double gloves and sleeve covers. 4. Remove the seal from the preamp plate and carefully remove and discard the outer gloves. Dilute the preamp products at 1:5 by adding 56 µl of TE buffer to each well of columns 1-6 of the 96-well preamp plate and mixing by pipetting up and down. Go only to the first stop of the pipette, aspirating from the bottom and dispensing higher up but not creating bubbles. Add TE buffer to all 6 columns regardless of unused wells. 5. Reseal the plate with either a clear adhesive or foil seal, and centrifuge it for 20 sec using the PCR plate spinner. Set this plate aside (loosely covered) until step 6.1 is complete. Discard remaining gloves and sleeve covers. box cover when a new box is opened, and ensure that the tips are not expired. Turn on the liquid handler and computer and open the liquid handler software, which will perform a self-test. 3. Click "Setup and Load". Click "Browse" to select the csv file created from the Sample Tracker software. If the file is not shown, go to "Instrument/Edit Preferences" and then click "Change" by "Sample Plate File Folder". Select the folder where the csv file is saved. Then, click "Setup and Load", then "Browse" and select the file. 4. Next, click "Browse" next to the "Plate Holder Position 1" and select the TPF file (provided by the manufacturer) that has the same serial number as the plate.