RNA extraction

Lyse cells with 1ml Trizol (Invitrogen) and pipette several times in the fume hood
Transfer to eppies
Add 0.2ml chloroform per 1ml Trizol in fume hood
Shake by hand vigorously for 15s
Let stand 5 min
Spin 12000 rcf for 15min, 4C
Transfer upper phase (clear) to new tube (~380ul). Don’t touch interface
Add equal volume of 70% EtOH (EtOH in RNA grade water) to collected samples.
Mix.
Add sample to RNeasy kit column (~700ul)
Spin 10 000 rpm, 30s, discard flowthrough
Add 700ul Buffer RW1 (from Qiagen RNeasy kit)
Add 500ul Buffer RPE
Spin 10 000rpm, 30s, discard flowthrough
Add another 500ul Buffer RPE
Spin 10 000rpm, 30s, discard flowthrough
Centrifuge full speed 1min
Elute RNA with 30-50ul of water
Centrifuge 10 000rpm, 1min
Nanodrop RNA (A260)