Set up qPCR with Cv_CatL_F/R primers. Plate layout/PCR set up is here.
Results: Waters are clean. 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.
Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for gDNA still. These were retreated according to Ambion protocol with a brand new Turbo DNA-free DNase kit. Additionally, I tested all three existing kits by “spiking” 19uL of H2O with 1uL (~200ng) of gigas gDNA; one tube for each kit and an untreated sample. Will qPCR to see if gDNA removal was successful.
The entire 20uL of RNA were treated with Ambion’s Turbo DNA-free kit according to protocol and spec’d.
Mac performed qPCR on the DNase-treated RNAs to verify removal of residual gDNA.
Results: Samples 3326: B23, A25, A22, B14, A21, A10 B22 came up positive for residual gDNA. Will retreat these samples and qPCR again.
Samples were pelleted and washed with 70% EtOH according to Ambion PolyA Purist protocol. Pellets were resuspended in 10uL of The RNA Storage Solution (included in the Ambion PolyA Purist Kit).
Results: The gill mRNA looks great! Good yield and good ratios. Hemocyte mRNA looks kinda rough and a very low yield (which was to be expected).