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:

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