Set up qPCR with Cv_CIAPIN_F/R primers. Plate layout/PCR set up is here.
Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.
Set up qPCR with Cv_CatY_F/R primers. Plate layout/PCR set up is here.
Results: Waters are clean. gDNA samples did not amplify. To be analyzed later with other genes that Mac has run.
DNase treated the 07:12 set of Abalone Dg RNA. Also, respec’d the RNA prior to proceeding. Followed Ambion’s Turbo DNA-free Kit rigorous protocol. Here are the calcs/workup for the DNase treatment. This used previously untreated RNA.
Due to the excessive number of times that these samples have been DNased, I’m concerned that the buffer is becoming too concentrated. So, I’m performing an EtOH precip on them to clean them up and then proceed with another round of DNase treatment.
0.1 volumes of 3M sodium acetate (pH = 5.2) were added to each sample. Then, 2 volumes of 100%, ice-cold EtOH were added. Samples were mixed and stored @ -20C for 2hrs. Samples were then centrifuged @ 4C, 16,000g for 30mins. Supe removed. Added 1mL of 70% EtOH and then centrifuged samples @ 4C, 16,000g for 15mins. Supe was removed, tubes were spun briefly to pool residual EtOH and the remaining EtOH was removed. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d.
Results: The following samples appear to have no RNA after precipitation:
06:5-34, 37; 06:6-44, 45, 46, 47, 52; 08:4-9, 10, 13, 15-18; All of the 07:12 samples.
Since I did not DNase the 07:12 samples, I can’t say whether or not this result was expected (e.g. due to low initial concentration). The others that have no remaining RNA are a surprise and is a bit disconcerting.
Will take fresh RNA aliquots of those samples for DNasing. For those samples still having RNA post-recip, I will just use them as they are, as most concentrations are in the recommended range for DNasing, according to the Ambion Tubro DNA-free kit.
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.
Set up qPCR using H.crach_16s_SYBR_F/R primers. Plate layout/set up is here.
Results: Waters are clean, but fluorescence still coming up in the samples!
This is the 4th time this RNA has been DNased. Performed using Ambion’s Tubro DNA-free Kit. Followed the “rigorous” treatment protocol.
Set up qPCR with Cv_TLR _short_F/R primers. Plate layout/PCR set up is here.
Results: Waters are clean. Melting curves look a bit rough, though. To be analyzed later with other genes that Mac has run.
Set up qPCR with Cv_BgBL_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.
