Changes

*RNeasy PureLink RNA Mini Kit (Invitrogencat#12183-018A)*Mouse Tissue (2050-30 100 mg, about a 3mm cube)*TRIZol (Invitrogen cat# 12183-555)*Chloroform (in solvent cabinet)*Label tubes, for each sample need 2 3 eppies, at least one RNeasyof which is 2 mL and one of which is a recovery tube from the PureLink kit. 

#Cut ~50-100 mg of tissue and weigh . If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube to ensure a sample of up to 30 mg tissueand keep on dry ice (if frozen).#Prepare buffer RLT by adding 10 uL B-ME per Add 1 mL of RLT in a clean 15 mL falcon TRIzol reagent to each tube. Need 600 uL per sample#Using dounce homogenizertissue grinder, homogenize tissue 10x and transfer to a clean tubefor ~30s until homogeneous (no clumps).#Centrifuge 3 min Incubate 5 minutes at room temperature .#Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.#Incubate at 14 000 RPMroom temperature for 2-3 minutes.#Remove 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 600 uL of the upper phase to a clean fresh tube.#Add 600 uL of 70% ethanol and mix immediately by pipettingvortexing.#Remove 700 uL of mixture (a precipitate may have formedinvert briefly to mix before taking sample) and add to a RNeasy spin column in a collection tube.#Spin 15s at 14 000 RPMon max (press button). Discard flow through. Add remaining sample and respin.#Add 700 uL RW1 Wash Buffer I to spin columcolumn.#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) Spin 15s on max. Discard flow through and mixthe collection tube. Get a new collection tube.#Add this mixture (80 500 uL per sampleWash Buffer II (make sure ethanol was added to the wash buffer) to the spin columns and sit cartridge.#Spin 15s on bench for 15 minmax. Discard the flow through and replace the collection tube.#Add 350 Repeat wash by adding 500 uL RW1 Wash Buffer II to the spin columncartridge.#Spin 15s at 14 000 RPMon max. Discard the flow through#Add 500 uL RPEand keep the same collection tube.#Spin 2 1 min at 14 000 RPM#Remove spin column on max to a new eppie dry the cartridge. Discard the collection tube and spin again to remove residual RPE#place into a clean recovery tube. Add 50 100 uL RNAse RNAase free water to the center of each tube. This can be adjusted to between 30 and spin 1 300 uL elution buffer if necessary. #Incubate at room temperature for 1min.#Spin 2 min on max to elute get purified RNA.#Quantify the RNA using the nanodrop.