RNA Extraction from axolotl tissue: Difference between revisions

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<h3>Materials:</h3>
== Material ==
<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
 
== 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

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:

  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
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