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.
Transferred 50uL of RNA to fresh tubes and processed them using the Ambion Turbo DNA-free kit according to the manufacturer’s protocol.
Anneal 55C. PCR set up is here (bottom half of sheet).
Lane 1 – Hyperladder
Lane 2 – gDNA
Lane 3 – cDNA pool
Lane 4 – No RT 06:5-31
Lane 5 – No RT 06:6-43
Lane 6 – No RT 08:3-5
Lane 7 – No RT 08:3-6
Lane 8 – No RT 08:3-7
Lane 9 – H2O
Lane 10 – H2O
Lane 11 – 100bp ladder
Results: No signal in the gDNA. Appropriate sized band in cDNA pool. Nothing in the water samples. HOWEVER, got bands in tow of the “No RT” rxns (08:3-6/7)!! It’s odd that the gDNA didn’t produce any signal, but there shouldn’t be any signal in the No RT rxns. This indicates gDNA contamination. Should have also tested RNA and DNased RNA. Will test these in order to determine if that system is better at eliminating gDNA carryover.
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.
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-6
08:3-7
08:3-8
08:3-9
08:3-14
08:3-15
08:3-16
08:3-24
Results: RNA looks great. Stored @ -80C in box where samples came from.
Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:
08:3-11
08:3-12
08:3-13
08:3-19
08:3-20
08:3-21
08:3-22
08:3-23
Notes: After phase separation, 3-11 and 3-12 had milky/cloudy aqueous phases. These two samples were subjected to another 10min spin @ 2500g, but this spin made no difference in their appearance. Other samples were clear or slightly translucent at worst.
Results: RNA looks great in nearly all of the samples. RNA has been stored @ -80C in the same box from where the tissue was taken.
