Due to craziness seen in melting curves, fluorescence, and empty wells from the previous run, will compare SYTO vs. SYBR with select MV cDNAs. Additionally, acquired some qPCR strip caps to use instead of the ABI film. Used Cv_18s_F/R primers. qPCR set up/plate layout is here.
Results: Both seem to work fine. H2O fluorescence is weird, but doesn’t come up in the melting curves. Strategene SYBR provides a brighter signal, but results in a higher melting temp than the SYTO.
Results: DNase treatment worked on all but the following samples: B23, B14, A21. However, these three samples were slightly below the initial, background fluorescence in each sample. The Turbo kit test indicates that all three kits are working perfectly and all can/should be used with confidence for treating samples.
Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.
Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA contamination. These will NOT be used to make cDNA for subsequent qPCRs.
Results: All samples produced a signal. In retrospect, this is likely due to having too much RNA for the DNase treatment. I proceeded with the DNase treatment and qPCR prior to specing the samples. Spec revealed that most of them were highly concentrated; more than the Ambion protocol recommends. Will redo the DNase treatment on a subset of the samples using the appropriate quantity of RNA.