Today I extracted RNA from four practice samples using Tri-reagent, and without lyophilizing (lyophilizer is out of commission again for now).
Tubes I used:
101-A –> a mistake sample
471-1 –> Day 26, uninfected, ambient
462-2 –> Day 26, infected, ambient
482-2 –> Day 26, infected, ambient
Protocol (from Tri-reagent protocol in Sam’s notebook)
- Added 1mL TriReagent to each tube
-
vortexed to mix/dissolve solute
- incubated 5mins at RT
- added 200uL of chloroform, pipet and then vortexed 15s to mix
- incubated at RT for 5mins
- centrifuged 15mins, 12,000g, 4˚C
- transferred aqueous phase to new tube
- added 500uL isopropanol to aqueous phase
- mixed, incubated at RT for 10mins
- centrifuged 8mins, 12,000g, at RT
- discarded supernatant
- added 1mL 75% ethanol
- centrifuged 5mins, 12,000g at RT
- discarded supernatant
- resuspended in 10uL of 0.1% DEPC-treated H2O.
Qubit Results (5ul of sample):
The extracted RNA tubes live in Rack 5, Column 4, Row 3
I did not do anything with the supernatant. I only process hemolymph pellet samples today.
Thoughts on the protocol:
- At step 14, tube 482 was really difficult to discard the supernatant. THere was stringy, clear material that was difficult to avoid, so I wasn’t able to discard a lot of the 75% ethanol. In the other tubes, however, the stringy stuff was more or less easier to avoid and I was able to remove almost all of the 75% ethanol before resuspending the sample in 0.1% DEPC-treated H20.
- The aqeous phase was really apparent with the Trizol, which I never saw when I used the RNAzol. Using the RNAzol always ended up being that the blue RNAzol was either at the top of the tube, the middle/bottom, or sometimes even throughout the tube. This, in all four tubes, created a clearly defined aqeous phase, which made removing the aqeous phase so so easy.
- Even though the Qubit results were pretty good, as I have learned from my RNAzol situation this summer, I cannot trust it until I look at the samples on the Bioanalyzer!!