Due to some weird anomolies seen during my previous qPCRs with the H.crach RNA/cDNA samples (positive controls produced good fluorescence when tested in Column 1 of the Opticon, but consistently failed to produce virtually any fluorescence when in Column 6 of the Opticon), I’ve decided to check the Opticon’s fluorescence detection.
qPCR setup/plate layout is here. I’ve made a master mix using Cg_HSP70_F/R primers designed by Mac. gDNA is BB #11 (0.49ug/uL) from 20090519. The gDNA will be added to the master mix. All wells will be tested.
Results: Unfortunately, this does not look as tight as it should. The Cts range from 23.9 - 26.6 (Cts from Opticon 2 with default threshold settings). This is nearly a 3 cycle difference which represents a nearly 10-fold difference in “expression.”