This should be the last time I run this for the time being. Re-running this with undiluted RLOv- samples to improve the melt curve resolution for better comparison to the RLOv+ melt curves.
All samples were run in duplicate.
See the qPCR Report (see Results below) for plate layout, cycling params, etc.
These results are interesting and I believe they are real, as opposed to the confusing/conflicting information I got from the previous two qPCRs from earlier today with the phage portal gene primers.
The poor/confusing results from the two previous qPCR attempts seem to have stemmed from low sample concentration. Using undiluted RLOv- samples in this run has resulted in clear, definitive data.
The melt curves are of a single peak in all RLOv+ samples.
The phage portal gene is NOT detected in RLOv- samples. However, it is present in RLOv+ samples and at significantly lower abundance than the RLOv DNA helicase (DNA helicase comes up at ~23 Cqs in samples that have been diluted 1:1000, while the phage portal gene comes up at ~28 Cqs in UNDILUTED samples). Alternatively, it is possible that the phage portal qPCR is less efficient and/or is experiencing some sort of inhibition; both seem unlikely, though.
Will discuss with Carolyn to see if she wants to go forward with cloning/sequencing and construction of a plasmid standard curve for the phage portal gene.
qPCR Amplification Plots (DNA helicase in green; Phage portal gene in blue)
qPCR Amplification Plots of Phage Portal Gene
qPCR Melt Curves of Phage Portal Gene