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.
Prepared cDNA using an equal amount of RNA from all samples (442.6ng). This amount was based on using the maximum allowable volume of RNA for the RT rxn AND the sample with the lowest [RNA] (08:3-20; 24.59ng/uL). cDNA was prepared according to the Promega MMLV RT recommendations. Here is the work up for the cDNA rxns. cDNA was stored @ -20C.
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.
