Difference between revisions of "RNA Extraction from axolotl tissue"

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<h3>Materials:</h3>
+
== Materials ==
<li>Trizol (Invitrogen)
+
* Trizol (Invitrogen)
<li>Chloroform
+
* Chloroform
<li>Isopropanol
+
* Isopropanol
<li>RNase free water
+
* RNase free water
<li>Potter homogenizer plus appropriate accessories (Stoessel und Pistille)
+
* Potter homogenizer plus appropriate accessories (Stoessel und Pistille)
<li>5 ml syringe
+
* 5 ml syringe
<li>25 guage needle
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* 25 guage needle
<li>Autoclaved Eppendorf tubes
+
* Autoclaved Eppendorf tubes
<li>75% ethanol in DEPC water
+
* 75% ethanol in DEPC water
<h3>Procedure to extract total RNA:</h3>
+
== 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

Revision as of 10:48, 27 March 2015

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