Performed analytical sensitivity assay validation using NcoI linearized p16RK7. Testing limit of detection by running 20 replicates each of 30 copies, 10 copies, 3 copies, 1 copy and 0 copies. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). This will be repeated two more times to have a final replicate count of 60 replicates of each copy number.
The positive control sample used was p16RK3 3e6 from 20120730.
qPCR Data File (CFX96)
qPCR Report (PDF)
Results look really good. Detection all the way down to three copies and even some detection at one copy, however I’m not sure how that will hold up statistically.
The Cq values for the run are here. Baseline threshold was set to 400 RFUs and cycle # 41 in order to be compatible with previous qPCR data generated by Nate for this assay. Will get data entered into spreadsheet for statistical analysis.