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==Materials==
*RNeasy Kit (Invitrogen)
*Mouse Tissue (20-30 mg, about a 3mm cube)
*Label tubes, for each sample need 2 eppies, one RNeasy
==Protocol==
#Cut tissue and weigh in a fresh tube to ensure a sample of up to 30 mg tissue
#Prepare buffer RLT by adding 10 uL B-ME per mL of RLT in a clean 15 mL falcon tube. Need 600 uL per sample
#Using dounce homogenizer, homogenize tissue 10x and transfer to a clean tube
#Centrifuge 3 min at room temperature at 14 000 RPM
#Remove centrifuge to a clean tube
#Add 600 uL of 70% ethanol and mix immediately by pipetting
#Remove 700 uL of mixture (a precipitate may have formed) and add to a RNeasy spin column in a collection tube
#Spin 15s at 14 000 RPM. Discard flow through
#Add 700 uL RW1 to spin colum
#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) and mix
#Add this mixture (80 uL per sample) to spin columns and sit on bench for 15 min
#Add 350 uL RW1 to spin column
#Spin 15s at 14 000 RPM. Discard flow through
#Add 500 uL RPE
#Spin 2 min at 14 000 RPM
#Remove spin column to a new eppie and spin again to remove residual RPE
#Add 50 uL RNAse free water and spin 1 min to elute RNA
*RNeasy Kit (Invitrogen)
*Mouse Tissue (20-30 mg, about a 3mm cube)
*Label tubes, for each sample need 2 eppies, one RNeasy
==Protocol==
#Cut tissue and weigh in a fresh tube to ensure a sample of up to 30 mg tissue
#Prepare buffer RLT by adding 10 uL B-ME per mL of RLT in a clean 15 mL falcon tube. Need 600 uL per sample
#Using dounce homogenizer, homogenize tissue 10x and transfer to a clean tube
#Centrifuge 3 min at room temperature at 14 000 RPM
#Remove centrifuge to a clean tube
#Add 600 uL of 70% ethanol and mix immediately by pipetting
#Remove 700 uL of mixture (a precipitate may have formed) and add to a RNeasy spin column in a collection tube
#Spin 15s at 14 000 RPM. Discard flow through
#Add 700 uL RW1 to spin colum
#Per sample, combine 10 uL DNAse I (in Enzymes Box) with 70 uL RDD (in Molecular Biology Box) and mix
#Add this mixture (80 uL per sample) to spin columns and sit on bench for 15 min
#Add 350 uL RW1 to spin column
#Spin 15s at 14 000 RPM. Discard flow through
#Add 500 uL RPE
#Spin 2 min at 14 000 RPM
#Remove spin column to a new eppie and spin again to remove residual RPE
#Add 50 uL RNAse free water and spin 1 min to elute RNA