RNA Extraction from axolotl tissue: Difference between revisions

Revision as of 08:49, 10 March 2014

Trizol (Invitrogen)

Chloroform

Isopropanol

RNase free water

Potter homogenizer plus appropriate accessories (Stoessel und Pistille)

5 ml syringe

25 guage needle

Autoclaved Eppendorf tubes

75% ethanol in DEPC water

Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer
Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue
Homogenize the tissue by passing through a syringe with a guage needle and incubate for 5 min @ Rt
Add .2 ml chloroform per ml of Trizol
Vortex for 15 sec
Incubate 5 min @ Rt
Centrifuge for 10 min @ 4 degree and 13000 rpm
Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix
Mix well
Incubate @ RT for 10 min
Centrifuge again @ 4 degree and 13,000 rpm
Remove the supernatant and wash the pellet in 75% and 100% Ethanol
Air dry the pellet and dissolve in RNase free water
Stores were kept in liquid nitrogen

@@ Line 10: / Line 10: @@
 <li>75% ethanol in DEPC water</li>
 <h3>Procedure to extract total RNA:</h3>
+# Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer
+# Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue
+# Homogenize the tissue by passing through a syringe with a guage needle and incubate for 5 min @ Rt
+# Add .2 ml chloroform per ml of Trizol
+# Vortex for 15 sec
+# Incubate 5 min @ Rt
+# Centrifuge for 10 min @ 4 degree and 13000 rpm
+# Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix
+# Mix well
+# Incubate @ RT for 10 min
+# Centrifuge again @ 4 degree and 13,000 rpm
+# Remove the supernatant and wash the pellet in 75% and 100% Ethanol
+# Air dry the pellet and dissolve in RNase free water
+# Stores were kept in liquid nitrogen