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.
Tag Archives: 08:4
RNA Isolation – Abalone Dg Project
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.
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.
gDNA Removal – Abalone RNA from 20090402 and 20090331
Transferred 50uL of RNA to fresh tubes and processed them using the Ambion Turbo DNA-free kit according to the manufacturer’s protocol.
cDNA – Abalone RNA from 20090331 & 20090402
cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a “No RT” rxn and thus, have duplicate wells (see plate layout).
The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.
The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).
UPDATE: cDNA plate was discarded 20120320 by SJW.
RNA Isolation – Abalone digestive gland samples
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-17
08:3-18
08:4-2
08:4-3
08:4-4
08:4-5
08:4-7
08:4-8
08:4-11
Notes: 08:4-5 took hours to wash the column. 08:4-4 ended up with a dark RNA pellet and the reuspended RNA is discolored (brown-ish).
Results: All the RNA looks good except for 08:4-4, but that isn’t surprising (see note above). Samples were stored @ -80C in the boxes from which the tissue samples came.