RNA extraction from axolotl tissue
Revision as of 09:21, 6 March 2014 by Stephanh (talk | contribs) (→Procedure to extract total RNA:)
RNA Extraction from axolotl tissue
Materials:
- 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
Procedure to extract total RNA:
-
- 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