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Rapid Isolation of RNA, DNA, and Proteins with Lumizol Reagent

Lumizol Reagent is designed for the rapid isolation of RNA, DNA, and proteins from cell and tissue samples of various origins (plants, animals, bacteria, and yeast). Lumizol Reagent is a solution containing phenol, guanidine isothiocyanate, and other components required for isolation of high-quality nucleic acids and proteins. Phenol and guanidine isothiocyanate provide cell lysis and effectively inhibit ribonucleases, thereby keeping RNA intact. After homogenizing and adding chloroform, the sample separates into an upper aqueous phase, interphase, and lower organic phase. The RNA can be precipitated with isopropanol from the upper phase, while the DNA and proteins can be precipitated with ethanol and isopropanol, respectively, from the interphase and lower organic phases.

Samples for RNA, DNA, and protein isolation:

  • plant and animal tissues — 30–100 mg;
  • adherent eukaryotic cells — 1×105 to 1×10;
  • suspension eukaryotic cells, yeast — 5×106 to 10×106;
  • bacterial cells — 1×107.

RNA isolation

Use fresh or liquid nitrogen snap-frozen samples for the best RNA isolation. Do not allow thawing of the samples before homogenization in Lumizol Reagent.

  1. Lyse samples in Lumizol Reagent according to your starting material:
    • Tissues: Homogenize the tissue sample in 1 mL of Lumizol Reagent and transfer the homogenate to a new 1.5 mL tube.
    • Cells: Discard the medium, resuspend the cell pellet or monolayer with 0.5–1 mL of Lumizol Reagent, and transfer the lysate to a new 1.5 mL tube.
  2. (Optional) If the sample has a high-fat content, centrifuge the lysate at 12,000 × g for 5 min at 4 °C and transfer the supernatant to a new 1.5 mL tube.
  3. Incubate the tube for 5 min at room temperature.
  4. Add 0.2 mL of chloroform to the lysate (per 1 mL of Lumizol Reagent used for lysis), mix thoroughly by inverting the tube, and incubate for 2 min at room temperature.
  5. Centrifuge the tube at 12,000 × g for 15 min at 4 °C. After centrifugation, the mixture separates into two phases: a colorless aqueous phase and a yellow organic phase, with an interphase between them.
  6. Transfer the upper aqueous phase to a new 1.5 mL tube without touching the interphase. If the amount of RNA in the sample is very small, add a coprecipitant to the aqueous phase. Keep the organic phase and interphase for DNA and protein extraction.
  7. Add 0.8 volumes of isopropanol to the aqueous phase, mix thoroughly, and incubate for 10 min at room temperature.
  8. Centrifuge the tube at 12,000 × g for 10 min at 4 °C.
  9. Discard the supernatant, add 1 mL of 70% ethanol to the RNA pellet, mix thoroughly, and centrifuge the tube at 7,500 × g for 5 min at 4 °C.
  10. Discard the supernatant and air dry the RNA pellet for 5–10 min.
  11. Dissolve the RNA pellet in 50–100 μL of RNase-free water.

DNA isolation

  1. Remove any remaining aqueous phase overlying the interphase obtained at step 5 in the «RNA isolation» section.
  2. Add 0.3 mL of 100% ethanol to the interphase and organic phase (per 1 mL of Lumizol Reagent used at step 1 in the «RNA isolation» section), mix thoroughly by inverting the tube and incubate for 2–3 min at room temperature.
  3. Centrifuge the tube at 2,000 × g for 5 min at 4 °C. Transfer the supernatant to a new 1.5 mL tube. The supernatant may be used for subsequent protein extraction.
  4. Resuspend the DNA pellet in 1 mL of sodium citrate/ethanol solution (0.1 M sodium citrate in 10% ethanol, pH 8.5).
  5. Incubate for 30 min, mixing occasionally.
  6. Centrifuge the tube at 2,000 × g for 5 min at 4 °C. Discard the supernatant.
  7. Repeat steps 4–6 one more time.
  8. Add 1 mL of 70% ethanol to the DNA pellet, mix thoroughly, and centrifuge the tube at 2,000 × g for 5 min at 4 °C.
  9. Discard the supernatant and air dry the DNA pellet for 5–10 min.
  10. Resuspend the DNA pellet in 0.3–0.6 mL of 8 mM sodium hydroxide.
  11. Centrifuge the tube at 12,000 × g for 10 min at 4 °C, transfer the supernatant to a new 1.5 mL tube, and adjust pH to 7–8 with Tris-HCl buffer [add 5 μL of 1 M Tris-HCl buffer (pH 4.5) to 300 μL of DNA solution].

Protein isolation

  1. Add 1.5 mL of isopropanol to the supernatant obtained at step 3 in the «DNA isolation» section (per 1 mL of Lumizol Reagent used at step 1 in the «RNA isolation» section), mix thoroughly by inverting the tube, and incubate for 10 min at room temperature.
  2. Centrifuge the tube at 12,000 × g for 10 min at 4 °C. Discard the supernatant.
  3. Resuspend the protein pellet in 2 mL of guanidine hydrochloride/ethanol solution (0.3 M guanidine hydrochloride in 95% ethanol).
  4. Incubate the tube for 20 min at room temperature.
  5. Centrifuge the tube at 7,500 × g for 5 min at 4 °C. Discard the supernatant.
  6. Repeat steps 3–5 two more times.
  7. Add 2 mL of 100% ethanol to the protein pellet, mix thoroughly, and incubate for 20 min at room temperature.
  8. Centrifuge the tube at 7,500 × g for 5 min at 4 °C. Discard the supernatant.
  9. Air dry the protein pellet for 5–10 min.
  10. Dissolve the protein pellet in a SDS-containing buffer (e.g., a sample buffer for loading protein samples onto a polyacrylamide gel).
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