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→Materials
==Safety Information==
* [[SOP-_Phenol|SOP - Phenol]]
* [[SOP_-_Chloroform|SOP - Chloroform]]
==Materials==
*PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
*TRIZol (Invitrogen cat# 12183-555)
*Chloroform (in solvent cabinet)
*Label tubes, for each sample need 3 eppies, at least one of which is : 2 .0 mL and one of which is a tube (to cut sample into), 1.5 mL tubes (X2), 1 spin column*, 1 recovery tube from *, 1 collection tube* (*= in the PureLink kit.)
*70% Ethanol make with RNAase free water and 100% Ethanol.
==Protocol==
#Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen, cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice.
#Add 1 mL TRIzol reagent to each 2 mL tube.
#Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf Add 1 ball bearing to each tube and keep on ice.#Using tissue grinder, homogenize tissue for ~30s until homogeneous 3 minutes at 25 Hz (make sure there are no remaining clumps, maybe take longer for muscle). If chunks remain after 3 minutes, homogenize until chunks are gone in 2 minute increments.
#Incubate 5 minutes at room temperature.
#Transfer sample to a new 1.5 vial. The ball bearings are reusable after cleaning so do not discard them.#Add 200 uL Chloroform and shake vigourously by hand for 15s15 seconds. Do not NOT vortex.
#Incubate at room temperature for 2-3 minutes.
#Centrifuge in a cold eppendorf centrifuge (4C) on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA. (WAT tissue may separate into 3 phases, RNA is the top, colorless phase.#Transfer 600 Add 400 uL of the upper phase 70% ethanol to a fresh tube.#Add 600 Carefully transfer 400 uL of 70% the upper phase to the ethanol tube and mix by vortexing. The remaining chloroform in the previous vial should be disposed of in the phenol waste container under the hood.#Remove 700 uL of ethanol/upper phase mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.#Spin 15s 15 seconds on max (press button). Discard flow through. Add remaining sample and , respinand discard flow through.
#Add 700 uL Wash Buffer I to spin column.
#Spin 15s 15 seconds on max. Discard flow through and the collection tube. Get , add a new collection tube.
#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
#Spin 15s 15 seconds on max. Discard the flow through and replace keep the same collection tube.
#Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
#Spin 15s 15 seconds on max. Discard the flow through and keep the same collection tube.#Spin 1 min minute on max to dry the cartridge. Discard the collection tube and place spin column into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary. #Incubate at room temperature for 1min1 minute.#Spin 2 min on max minutes at 12500 rpm to get purified RNA.
#Quantify the RNA using the nanodrop.
