Performed a dilution series to have the desired copy number using Low TE Buffer. The calculations were performed using a spreadsheet developed by Nate. The calculations are here and the workbook has a separate sheet for each clone. qPCR master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Reports below (see Results).
qPCR Data File (CFX96)
SUCCESS!!! It’s a friggin’ miracle! Finally, we have three clones (pWC8 did not amplify at all!) that are useable for the withering syndrome assay!! Here are the r^2 values and the efficiency for each of the three clones that did work:
p16RK3 – r^2 = 0.996, E = 91.7%
p16RK7 – r^2 = 0.995, E = 98.4%
p18RK7 – r^2 = 0.998, E = 94.1%
Due to it having the best efficiency, p16RK7 clone will be used for the assay validation.
UPDATED 20121024 – Modified data file (and subsequently the qPCR Report) to have a baseline threshold of 400 and cycles to analyze 41 to match existing conditions used for the withering syndrome qPCR assay validation. HOWEVER, I have NOT updated the r^2 and efficiency numbers listed above to reflect the new baseline setting! Please see the respective qPCR Reports for that information!!