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→Protocol
#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 tube and keep on ice.
#Using tissue grinder, homogenize tissue for ~30s until homogeneous 3 minutes at 25Hz (make sure there are no remaining clumps).
#Incubate 5 minutes at room temperature.
#Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.
#Incubate at room temperature for 2-3 minutes.
#Centrifuge in a cold eppendorf centrifuge 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.
#Transfer Add 400 uL of the upper phase 70% ethanol to a fresh tube.#Add Transfer 400 uL of 70% the upper phase to the ethanol tube and mix by vortexing.
#Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
#Spin 15s on max (press button). Discard flow through. Add remaining sample and respin.
#Add 700 uL Wash Buffer I to spin column.
#Spin 15s on max. Discard flow through and the collection tube. <s>Get a new collection tube.</s>
#Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
#Spin 15s on max. Discard the flow through and replace the collection tube.
#Spin 1 min on max to dry the cartridge. Discard the collection tube and place 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 1min.
#Spin 2 min on max 12500 rpm to get purified RNA.
#Quantify the RNA using the nanodrop.
