RNA extraction from axolotl tissue: Difference between revisions

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(Created page with "== RNA Extraction from axolotl tissue == === Materials: === <ul> <li>Trizol (Invitrogen)</li> <li>Chloroform</li> <li>Isopropanol</li> <li>RNase free water</li> <...")
 
 
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=== Procedure to extract total RNA: ===
=== Procedure to extract total RNA: ===
<ol>
<ol>
<li>
     <li>Frozen tissue (measure the weight before starting), grinded to a powder in liquid nitrogen with the homogenizer</li>
  <ol style="list-style-type:lower-roman">
    <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>
- Add 1 ml Trizol to 50 – 100 mg of deep frozen tissue
    <li>Add ,.2 ml chloroform per ml of Trizol</li>
- Homogenize the tissue by passing through a syringe with a guage needle and incubate for 5 min @ Rt
    <li>Vortex for 15 sec</li>
- Add ,.2 ml chloroform per ml of Trizol
    <li>Incubate 5 min @ Rt</li>
- Vortex for 15 sec  
    <li>Centrifuge for 10 min @ 4 degree and 13000 rpm</li>
- Incubate 5 min @ Rt
    <li>Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix</li>
- Centrifuge for 10 min @ 4 degree and 13000 rpm
    <li>Mix well</li>
- Take the aquerious phase and there was added 0.25 ml of isopropanol and 0.25 ml of RNA precipitation mix
    <li>Incubate @ RT for 10 min</li>
- Mix well
    <li>Centrifuge again @ 4 degree and 13,000 rpm</li>
- Incubate @ RT for 10 min
    <li>Remove the supernatant and wash the pellet in 75% and 100% Ethanol</li>
- Centrifuge again @ 4 degree and 13,000 rpm
    <li>Air dry the pellet and dissolve in RNase free water</li>
- Remove the supernatant and wash the pellet in 75% and 100% Ethanol
    <li>Stores were kept in liquid nitrogen</li>
- Air dry the pellet and dissolve in RNase free water
</ol>
- Stores were kept in liquid nitrogen

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:

  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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