Team:Evry/Notebook/Protocols/RNAextraction

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RNA isolation (using TRI Reagent)

Before : -Cleaning everything with RNA zap -Switch on centrifuge for a fast cool at 4C *Preparation of stabilisation buffer : Add to 15 mL facon tube : _ 5ml Phenol _ 5ml 1M sodium acetate pH=5.5 Centrifuge 3 min, 4k rpm Tranfer lower phase (contains phenol) to 50 mL falcon Transfer 2.5mL to second falcon (devide into 2 tubes) Add 45mL 100% ethanol to each tube *Preparation of the samples measurement of OD if log phase, centrifugation of culture Discard the supernatant *Stabilization of samples Add 1.25mL stabilization buffer to 10ml log phase culture Put culture on ice Vortex Transfer into 15 ml falcon tube Spin 4k rpm , 5 min Discard supernatant resuspend pellet in 1ml TRI Reagent transfer to 2 ml eppie incubate 5 min at RT add 200µL chloroform vortex 15 sec incubate 15min at RT Spin 12K rpm, 15min, 4C transfer clear, aqueous phase (upper phase) to fresh tube add 500µL isopropanol vortex 5 sec incubate 10' at RT spin 14 000, 10 mn, 4C carrefully pout off supernatant add 1 mL 75% ethanol spin 14000 rpm, 5min 4C discard ethanol air dry pellet 30 min Put 93µL RNAse free water Verification on an electrophoresis gel ( attention : preparation of electrophoresis gel with TBE or TAE RNAse Free agarose 1%) Measuring quantity of RNA with nanodrop Turbo DNAse protocol of RNA Dilute sample to 200ng/µL in 100µL (20µg total) Add 10µL 10X DNAse buffer Add 2µL turbo Dnase (2U/µL) Incubate at 37C, 30min *Phenol-Chloroform extraction Ajust the volume of this sample at 200µL Add 1 volume of phonol:chloroform:isomyl alcohol Vortex spin 14K , 5min, 4C Transfer upper , aqueous phase to fresh tube Verification on an electrophoresis gel (if DNA IS ALWAYS present, do again the step of Turbo DNAse treatment) *Protocol for ethanol/acetate precipitation of RNA if small volume, bring to 180µL with RNAse-free water add 0.1 volume 5M ammonium acetate or 3M sodium acetate (optional) add 2µL of 5µg/µL glycogen if RNA is < 200µg/µL add 2.5-3 volumes 100% ethanol put at -20C o/n or -80C for 30min 13K, 30min, 4C carrefully discard supernatant add 1ml ice cold 70% ethanol 13K, 10min, 4C discard ethanol air dry 10min Resuspend RNA in RNAse-free water (max 18µL) Verification on an electrophoresis gel *Nanodrop *RNAship