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!
Tag Archives: digestive gland
DNase Treatment – Abalone Dg DNased RNA 20090610
This is the 4th time this RNA has been DNased. Performed using Ambion’s Tubro DNA-free Kit. Followed the “rigorous” treatment protocol.
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.
DNase Treatment – Abalone Dg DNased RNA from 20090605
This is the 3rd time this will be performed on these samples. Hopefully using a different (i.e. new) kit will work. Not sure what’s going on here. Performed according to protocol.
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.
DNase Treatment – Abalone Dg DNased RNA from 20090605
Had to re-treat these samples due to residual gDNA that was NOT eliminated the first time through. Possible old Ambion kit??
qPCR – 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: RNA is still contaminated. Will have to re-DNase the samples.
DNase Treatment – Abalone Dg RNA isolated yesterday and from 20090518
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here.
RNA Isolation – Abalone Dg Project Samples
RNA was isolated from the following samples using the MoBio RNA PowerSoil Kit according to protocol:
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.