Today I tried out the Trizol LS Reagent extraction, but the centrifuge in the 4˚C in FTR 213 had an “Err 3” message, which happens when there’s an error in the rotational speed measurement system (GitHub Issue #542). I’ll put a different centrifuge in the 4C and re-try the protocol on Wednesday. This post includes my protocol for the Trizol LS Reagent extraction.
Trizol LS Reagent extraction
As stated in the blurb above, the centrifuge in the 4C fridge in FTR 213 has an error that won’t allow it to run. I’ll go through the protocol again on Wednesday (I have no classes on Wednesdays), run the samples on the Qubit, and run on the Bioanalyzer with the 8 Qiagen RNeasy Kit-extracted samples (Post from Nov 21, 2018).
The samples I started working with today (and which have been dumped due to the centrifuge error) are from Day 26. They are each the second out of the three samples that were taken for each crab. In other words, there is only one sample tube remaining for each of these three crabs. The tubes used today were: 427, 430, 433.
Trizol LS Reagent Extraction Protocol (I’ll be doing this on Wednesday):
Lyse samples and separate phases:
- Add 750uL Trizol LS Reagent to the samples (pelleted C. bairdi hemolymph)
(Need to maintain a 3:1 ratio of Trizol to sample. I estimated that the samples are about 250ul)
- Homogenize sample by pipetting (5x per sample)
- Centrifuge 5 mins at 12000g at 4C
- Transfer clear supernatant to a new, labeled tube
- Incubate 5 mins to permit complete dissociation of the nucleoproteins complex.
- Add 200ul of chloroform and close lid
- Incubate at room T 2-3 mins
- Centrifuge 15mins at 12000g at 4C
(The mixture separates into a lower phenol-chloroform, and interphase, and a colorless upper aqueous phase)
- Transfer aqueous phase containing RNA to a new tube by angling at 45˚ and pipetting out.
(DO NOT transfer any of inerphase or organic layer. ONLY the aqueous phase)
A. Precipitate RNA
- Add 500ul of ispropanol to the aqueous phase
- Incubate at room T 10 mins
- Centrifuge 10 mins at 12000g at 4C
(Total RNA becomes a white gel-like pellet at the bottom of the tube)
- Discard supernatant
B. Wash the RNA
- Resuspend pellet in 1mL of 75% ethanol
- Vortex briefly. Centrifuge for 5 mins at 7500g at 4C
- Discard supernatant
- Air dry pellet 5-10 mins
C. Solubilize the RNA
- Resuspend pellet in 20ul of 0.1% DEPC-treated H20
- Incubate in a heat block set to 55˚C for 15mins