Bead Beating: A Primer (continued)

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APPENDIX C

RNA Isolation from Plants

In addition to RNases, polyphenolics and polysaccharides compound the difficulty of isolating RNA from plants. Consequently a combination of methods is used to protect the RNA while eliminating contaminants.  RNA isolation is best accomplished using cryogenic homogenization.

Homogenization Buffer: 0.25 M NaCl, 50 mM Tris (pH 7.5), 20 mM EDTA, 1% SDS, 4% PVP

Isolation Reagents: Phenol saturated (stored under) with 42 mM citrate buffer, pH 4.3
chloroform/isoamyl alcohol (ratio of 24:1,
3 M sodium acetate, pH 5.2,
95% ethanol (store at -20°C),
70% ethanol (store at -20°C), and
water (molecular biology grade).
NOTE: Phenol can cause chemical burns and chloroform is carcinogenic. Exercise caution when working with these chemicals.

Procedure

  1. Plant tissue should be flash frozen when harvested and placed in a polycarbonate grinding vial for cryogenic processing.  Homogenize the sample cryogenically.   Do not allow the sample to thaw.
  2. Using a nuclease-free tube, add 750 µl of Homogenization Buffer and 750 µl chloroform/isoamyl alcohol for each 100 mg of homogenized plant tissue.  Add the powdered plant tissue and vortex for 30 seconds.
  3. Centrifuge the tube at 12,000 g to separate the phases.  Transfer the aqueous phase (upper) to a clean tube.
  4. Add an equal volume of buffer saturated phenol.  Vortex for 30 seconds and centrifuge to separate the phases.   Transfer the aqueous phase (upper) to a clean tube.
  5. Repeat the phenol extraction and transfer the supernatant to a clean tube.
  6. Add an equal volume of chloroform/isoamyl alcohol.  Vortex, centrifuge, and transfer supernatant to a clean tube.  Repeat this step.
  7. Add 1/10 volume of 3 M sodium acetate and mix.  Add 2.5 volumes of cold 95% ethanol.  Mix and incubate at -20°C for 30 minutes.
  8. Centrifuge the sample at 12,000 g for 20 minutes.  Decant and wash the pellet with cold 70% ethanol. Decant and air-dry the pellet.
  9. Dissolve the pellet in molecular biology grade water.  Store at -80°C.

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