RNA extraction from axolotl tissue: Difference between revisions
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=== Procedure to extract total RNA: === | === Procedure to extract total RNA: === | ||
<ol> | <ol> | ||
<li>Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer</li> | |||
<li>Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue</li> | |||
<li>Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer | <li>Homogenize the tissue by passing through a syringe with a guage needle and incubate for 5 min @ Rt</li> | ||
<li>Add ,.2 ml chloroform per ml of Trizol</li> | |||
<li>Vortex for 15 sec</li> | |||
<li>Incubate 5 min @ Rt</li> | |||
<li>Centrifuge for 10 min @ 4 degree and 13000 rpm</li> | |||
<li>Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix</li> | |||
<li>Mix well</li> | |||
<li>Incubate @ RT for 10 min</li> | |||
<li>Centrifuge again @ 4 degree and 13,000 rpm</li> | |||
<li>Remove the supernatant and wash the pellet in 75% and 100% Ethanol</li> | |||
<li>Air dry the pellet and dissolve in RNase free water</li> | |||
<li>Stores were kept in liquid nitrogen</li> | |||
</li> | |||
</ol> | </ol> | ||
Latest revision as of 09:30, 6 March 2014
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