Preparation of RNA Samples from Mouse Tissues: Difference between revisions
m Added where to dispose of the chloroform in case someone forgets. |
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#Add 1 mL TRIzol reagent to each 2 mL tube. | #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. | #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, and after adding the ball bearings to every vial, homogenize tissue for 3 min at 25Hz (make sure there are no remaining clumps). | #Using tissue grinder, and after adding the ball bearings to every vial, homogenize tissue for 3 min at 25Hz (make sure there are no remaining clumps, maybe take longer for muscle). | ||
#Incubate 5 minutes at room temperature. | #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 15s. Do not vortex. | #Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex. | ||
#Incubate at room temperature for 2-3 minutes. | #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. | #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. | ||