Difference between revisions of "RNA Extraction from axolotl tissue"
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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
Line 1: | Line 1: | ||
<h3>Materials:</h3> | <h3>Materials:</h3> | ||
− | <li>Trizol (Invitrogen) | + | <li>Trizol (Invitrogen) |
− | <li>Chloroform | + | <li>Chloroform |
− | <li>Isopropanol | + | <li>Isopropanol |
− | <li>RNase free water | + | <li>RNase free water |
− | <li>Potter homogenizer plus appropriate accessories (Stoessel und Pistille) | + | <li>Potter homogenizer plus appropriate accessories (Stoessel und Pistille) |
− | <li>5 ml syringe | + | <li>5 ml syringe |
− | <li>25 guage needle | + | <li>25 guage needle |
− | <li>Autoclaved Eppendorf tubes | + | <li>Autoclaved Eppendorf tubes |
− | <li>75% ethanol in DEPC water | + | <li>75% ethanol in DEPC water |
<h3>Procedure to extract total RNA:</h3> | <h3>Procedure to extract total RNA:</h3> | ||
# 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 |
Revision as of 08:52, 10 March 2014
Materials:
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