Bead Beating: A Primer (continued)
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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.
|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.|