Tag Archives: SYTO13

qPCR – MV hemocyte cDNA from yesterday

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 – DNased MV hemocyte RNA from earlier today AND Turbo kit test

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.

qPCR – Re-DNased abalone Dg RNA from earlier today

This is a repeat of the previous qPCR from earlier today, BUT I think I might have used the wrong primers in the earlier qPCR (see below). Set up qPCR with the correct (I’m 100% sure of this) primers. Plate layout/workup is here.

Results: Well, in retrospect it looks like I DID use the correct primers earlier! However, the problem is the same. But, the melting curves in the H2O-only samples don’t seem to be the same as what is being seen in the RNA samples, suggesting that the signal in the H2O-only samples are likely primer dimers (melting curve peaks are shifted to the left and are VERY low signals; barely above background).

So, what to do now? Mac has a mad ea suggestion to spike some water with gDNA and DNase treat the sample to assess whether or not the Dase treatment is actually working or not. I think I’ll do this.

qPCR – Re-DNased abalone Dg RNA from earlier today

Set up qPCR. Plate layout/workup is here.

Results: Looks like gDNA contamination is still present!! This is insane! However, the two water-only samples produced a signal suggesting that something else is contaminated. Will try just qPCR-ing water to see if I can get a clean signal. Will use “store-bought” PCR water instead of NanoPure water.

*UPDATE**: Possibly used 16s universal bacterial primers instead of H.crach 16s primers! Doh! Will re-qPCR using the correct primers.

qPCR – Re-DNased abalone Dg RNA from earlier today

Done to verify removal of gDNA from RNA. Used H.crach_16s_syb_f/r primers. PCR workup/plate layout is here.

Results: Still f’ing gDNA! I’m pretty convinced that this is indeed due to the Ambion kit I’m using being old. Got mixed up with a newer kit, but neither had dates. Mac is going to be running a qPCR later today on DNased RNA that used the other Ambion kit. I will wait until the results of her qPCR to proceed.

qPCR – Mac’s gigas DNased RNA from earlier today

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.

qPCR – Re-DNased oyster RNA from today

Performed qPCR on the re-DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the initial qPCR from yesterday indicated residual gDNA was still present in the DNase treated RNA. Plate layout/set up can be found here.

Results: About 4 samples in each site set are NEGATIVE for gDNA. That means the remainder still have detectable levels of gDNA. Boo.

qPCR – DNased oyster RNA from earlier today

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.

qPCR – Abalone DNased RNA from yesterday

Performed qPCR to evaluate gDNA removal w/ 2x Immomx and SYTO 13. qPCR/plate set up is here.

Results: The two cDNA samples come up as positive. No flourescence detected in any other gamples. However, melting curves look suspicous despite the fact that the “Quantitation” view indicates now amplification.