I previously ran three qPCR plates (20160121, 20160122, & 20160125) with 20 reps of each of the following copy numbers to determine the limit of detection (or, analytical sensitivity) of this qPCR assay: 30, 10, 3, 1
Additionally, I determined the average baseline threshold to set (580.5), based on eight qPCRs (see 20160128).
Performed calculations and determined limit of detection (defined as >95% of samples produced amplification) for this assay is three copies. The single copy sample amplified 88% of the time. This also means that our cycle threshold cut-off for this qPCR assay should be 37.9, as the mean Cq for three copies was 37.33, with a standard deviation of 0.53.
The data and calculations can be seen below (Google Sheet – scroll to the right to see calcs):
Google Sheet: RLOv_DNA_helicase_LoD_calcs
Idetermined a qPCR baseline threshold to use for the withering syndrome RLOv DNA helicase qPCR assay in the following fashion.
I adjusted baseline threshold values of the following eight qPCR runs so that the mean Cq values for each component of the standard curves (per plate) across plates were within 0.5 Cqs and averaged the baseline thresholds.
The average baseline threshold was 580.5.
This value will be manually applied to all past and future RLOv DNA helicase qPCR runs that were conducted on the Friedman Lab CFX96.
Ran limit of detection qPCR for the Promega 2x GoTaq Probe Master Mix using p18RK7 (from 20120730) primary curve and p18RK7 low curve (from 20140507).
Master mix calcs are here: 20140506 – qPCR WSN1 Promega LoD-1
Cycling params, plate layout, etc. can be found in the qPCR Report in the Results below.
qPCR Report (PDF): Sam_2014-05-21 14-04-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-21 14-04-07_CC009827.pcrd
Data can be found here: Promega Master Mix Withering Syndrome Limit of Detection