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.
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.
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.
Had to re-treat these samples due to residual gDNA that was NOT eliminated the first time through. Possible old Ambion kit??
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.
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here.
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.
RNA from yesterday was treated according to Ambion Turbo DNA-free protocol to remove gDNA contamination. Work up is here. Will check for residual gDNA contamination once I’ve finished with another set of RNA isolations from the remainder of Abalone Dg tissue.
Isolated RNA using the MoBio RNA PowerSoil Kit according to protocol from the following samples:
06:6-45
06:6-49
06:6-50
06:6-51
06:6-52
08:4-6
08:4-9
08:4-10
Results: RNA looks fabulous (as always from the PowerSoil kit). Will DNase treat the samples tomorrow.
Isolated RNA from Abalone Dg samples (see below) using MoBio RNA PowerSoil Kit according to protocol. RNA was stored @ -80C.
Results: RNA quality looks great, as usual. Sample 06:6-44 has a very low yield, but was to be expected due to very small amount of starting tissue.
