Set up qPCR with Cv_18s_F/R primers on the following samples that had previously come up negative in both reps using the diluted (1:20) cDNA that Mac had made specifically for the 18s runs:
3326A13
2100B07
3219A06
2100B15
3326A11
2100B12
2100A03
Plate layout/PCR set up is here. This is a second rep of these samples.
Results: Waters are clean. However, the following samples still are negative for an 18s amplicon:
Set up qPCR with Cv_18s_F/R primers on the following samples that had previously come up negative in both reps using the diluted (1:20) cDNA that Mac had made specifically for the 18s runs:
3326A13
2100B07
3219A06
2100B15
3326A11
2100B12
2100A03
Plate layout/PCR set up is here.
Results: Waters are clean. However, the following samples still are negative for an 18s amplicon:
Set up qPCR with Cv_18s_F/R primers. Plate layout/PCR set up is here. This is a second rep. Used 1:20 cDNA plate made by Mac as template.
Results: Waters are clean. Still some samples that aren’t coming up. Will eventually run those samples using undiluted cDNA. To be analyzed later with other genes that Mac has run.
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.
qPCR set up/plate layout is here. Used Cv_18s_F/R primers to assess samples’ “useability” for future qPCRs. Used an ABI optically clear adhesive film instead of caps. Ran out of appropriate caps.
Results: Yep, seal was bad. Explains most of the weirdness seen. However, will compare SYTO and Strategene SYBR.
qPCR set up/plate layout is here. Used Cv_18s_F/R primers for the MV hemocyte RNA and Gigas_18s_F/R primers for the Turbo kit test. Anneal 55C.
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.
