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Team:Evry/Notebook/Protocols/RNAextraction
From 2014.igem.org
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 (caution: 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
-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 (caution: 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
