Haynes:TRIzol RNeasy

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: <- [http://openwetware.org/wiki/Haynes:Protocols Back to Protocols] '''RNA Miniprep - TRIzol/ RNeasy Sping Column Combo'''<br> by Karmella Haynes, 2013 Principle: LYSE CELLS IN TRI...)
Line 11: Line 11:
LYSE CELLS IN TRIZOL
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''':  
# '''Adherent cells''':  
 +
EXTRACT RNA - PHASE SEPARATION
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.

Revision as of 14:45, 7 January 2013

<- Back to Protocols


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


Principle:


LYSE CELLS IN TRIZOL

  1. 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.
  2. Adherent cells:


EXTRACT RNA - PHASE SEPARATION

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