Chlamydomonas RNA extraction
RNA preparation This is a protocol designed to extract RNA from Chlamydomonas reinhardtii.
- Chlamydomonas in liquid nutrient medium (take at least one week to grow)
- Liquid Nitrogen
- Trizol (500 μL per sample)
- Chloroform (600 μL per sample)
- NaCl 5M (40 μL per sample)
- Isopropanol (500 μL per sample)
- Etanol 75% (8 mL per sample)
- Mili-q water
- To obtain the pellet transfer 1.5 mL of medium to an esterile eppendorf (X the number of samples you need) and centrifugue 2.30 minutes at 8000 rpm dispose rapidly of all the supernatant.
- Repeat previous step 5 more times, transfering only 1 mL now.
- immerse the tubes in the liquid nitrogen and swirl to cool.
- Add 500 μL Trizol (works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components) to each tube and then vortex.
- When the suspension is homogeneous, incubate for ten minutes at room temperature
- Add 600 μL Chloroform to each tube and shake vigorously for 1 minute. Incubate at room temperature for an additonal 2-3 minutes. You should see a separation of the aqueous and organic phases (organic at the bottom).
- Centrifugue 10 minutes at 12000 x g
- Separate the aqueous phase with extreme caution to avoid taking organic phase and transfer to new eppendorf tubes.
- Add 40 μL of NaCl 5M followed by the addition of 500 μL Isopropanol (this is to precipitate nucleic acids)
- Precipitation is performed at room temperature for 10 minutes. The precipitated nucleic acids are collected by 10 minutes-centrifugation at 12000 x g
- The nucleic acids are washed with ethanol 75%, dried (speed vac), and dissolved in Mili-q water.