Sean Lauber:RNA Extraction Using Trizol: Difference between revisions
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(New page: ===Procedure=== For extraction of RNA from cells on a 6-well plate) |
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===Procedure=== | ===Procedure=== | ||
#Ensure your cells are alive (if they're dead you won't get any RNA!) | |||
#Remove media on cells and add 1 ml of Trizol (regardless of plate size) | |||
<b> DO THIS ON THE BENCH AND NOT IN THE TISSUE CULTURE HOOD</b> | |||
#Scrape cells and pipette into a 1.5 ml eppendorf | |||
#Pipette up and down to lyse cells | |||
#Incubate at RT for 5 min | |||
<b>At this point you can store the sample at -80*C or continue</b> | |||
#In a fume hood, add 200 μL chloroform, shake to mix | |||
#Incubate at RT for 3 min | |||
#Spin at 13K RPM/4*C/15 min | |||
#Pipette 350 μL of the top (aqueous) layer into a new tube | |||
<b>You can remove more if you enjoy living dangerously but you risk contamination with DNA</b> | |||
Revision as of 11:44, 29 March 2012
Procedure
- Ensure your cells are alive (if they're dead you won't get any RNA!)
- Remove media on cells and add 1 ml of Trizol (regardless of plate size)
DO THIS ON THE BENCH AND NOT IN THE TISSUE CULTURE HOOD
- Scrape cells and pipette into a 1.5 ml eppendorf
- Pipette up and down to lyse cells
- Incubate at RT for 5 min
At this point you can store the sample at -80*C or continue
- In a fume hood, add 200 μL chloroform, shake to mix
- Incubate at RT for 3 min
- Spin at 13K RPM/4*C/15 min
- Pipette 350 μL of the top (aqueous) layer into a new tube
You can remove more if you enjoy living dangerously but you risk contamination with DNA
