Tag Archives: black abalone

qPCR – DNased Abalone Dg RNA from 20090625

Ran qPCR on DNased Abalone Dg RNA (07:12 Set), gDNA (06:50-10) and clean cDNA (from 20090422) using primers (H.crach_h-1fg_intron_Fw/Rv) designed to bind only to a region in an intron of the H.cracherodii hemocyanin gene. PCR setup/plate layout is here. Anneal temp of 50C was used.

Results: No PCR products in any samples, not even the positive control. It seems that the primers don’t work. Will design new primers, probably from a different species of abalone since there was essentially only one gene in H.cracherodii that had any intron sequence available..

qPCR – Abalone Dg DNased RNA from yesterday and earlier today

Check Abalone RNA for residual gDNA contamination after DNase treatment. Ran qPCR with H.crach_16s_SYB_F/R primers. Plate layout/PCR set up here.

Results: Still gDNA in virtually every sample! This is totally insane. Will find/design primers that will only amplify gDNA to aid in subsequent analysis…

EtOH Precipitation – DNased Abalone Dg RNA from yesterday AND the 07:12 set (DNased by Lisa 20090306)

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.

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.