Haynes:TRIzol RNeasy

(Difference between revisions)

Revision as of 14:45, 7 January 2013

RNA Miniprep - TRIzol/ RNeasy Sping Column Combo
by Karmella Haynes, 2013

Principle:

LYSE CELLS IN TRIZOL

Suspension cells: Add up to 1x10^6 cells to a 2.0 mL microfuge tube. Spin for 5 min. at 800 xg (~3000 rpm in a standard table top centrifuge) at room temp. Discard supernatant and add 500 μL TRIzol (per 1x10^6 cells). Mix by pipetting up and down. Incubate at room temperature for 5 min. Store at -80°C or go on to the next step.
Adherent cells:

EXTRACT RNA - PHASE SEPARATION

Add 200 μL chloroform per 1 mL TRIzol (100 μL per 500 μL TRIzol).
Make sure the lids are closed tightly. Mix by inverting for 20 seconds. Incubate at room temperature for 3 minutes.
Centrifuge samples at 12,000 xg for 15 minutes at 4°C.

@@ Line 11: / Line 11: @@
 LYSE CELLS IN TRIZOL
-# '''Suspension cells''': Add up to 1x10^6 cells to a 2.0 mL microfuge tube. Spin for 5 min. at 800 xg (~3000 rpm in a standard table top centrifuge) at room temp. Discard supernatant and add 500 m TRIzol (per . Mix by pipetting up and down. Incubate at room temperature for 5 min. Store at -80°C or go on to the next step.
+# '''Suspension cells''': Add up to 1x10^6 cells to a 2.0 mL microfuge tube. Spin for 5 min. at 800 xg (~3000 rpm in a standard table top centrifuge) at room temp. Discard supernatant and add 500 μL TRIzol (per 1x10^6 cells). Mix by pipetting up and down. Incubate at room temperature for 5 min. Store at -80°C or go on to the next step.
 # '''Adherent cells''':
 EXTRACT RNA - PHASE SEPARATION
-# Add 200 μL chloroform per 1 mL TRIzol (
+# Add 200 μL chloroform per 1 mL TRIzol (100 μL per 500 μL TRIzol).
+# Make sure the lids are closed '''tightly'''. Mix by inverting for 20 seconds. Incubate at room temperature for 3 minutes.
+# Centrifuge samples at 12,000 xg for 15 minutes at 4°C.