Performed qPCR using q18s primers on DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.
gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.
Results: gDNA dilutions look good. However, some samples are definitely coming up before the 40 cycle mark. Will re-DNase treat these.
Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params. Plate layout is here.
gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.
Results: Hello! I’m an idiot. Nothing amplified because the EF1 primers are designed to cross an intron/exon boundary, thus they can’t amplify gDNA. Need to use the 18s primers instead.
This is an exact duplicate of the earlier qPCR from today, but using the correct (18s) primers! Ran qPCR on RNA to evaluate gDNA contamination in the samples. Dilutions of the RNA were made at 1:100, which would be the equivalent amount when making cDNA (1:25) and diluting the cDNA (1:4) prior to using in a qPCR rxn. qPCR set up is here with cycling params except q18s primers are substituted instead of qEF1. Plate layout is here.
gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519.
Results: All samples show gDNA contamination. Will DNase them
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.
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.
Performed qPCR on the DNased RNA to with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples. Plate layout/set up can be found here.
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.
