RNA Extraction from axolotl tissue: Difference between revisions
Jump to navigation
Jump to search
No edit summary |
|||
| (One intermediate revision by the same user not shown) | |||
| Line 1: | Line 1: | ||
== Material == | |||
* 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 | # 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 | # Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue | ||
Latest revision as of 08:52, 19 June 2015
Material
- 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