Materials and reagents

Biological materials

1. Arabidopsis thaliana ecotype Columbia (Col-0) wild type (WT) and embryo-lethal mutant plants from the Arabidopsis biological resource center (ARBRC, https://abrc.osu.edu/)

Reagents

1. Tris [Tri(hydroxymethyl)aminomethane] (Genbiotech, catalog number: RU2510)

2. EDTA (ethylenediamine tetraacetic acid disodium salt) (Biopack, catalog number: 1604.07)

3. Urea (Sigma, catalog number: U5378)

4. Sodium dodecyl sulfate (SDS) (Anedra, catalog number: 7211)

5. Ammonium acetate (Fluka, catalog number: 09690)

6. Lithium chloride (LiCl) (Sigma, catalog number: L9650)

7. 2-Mercaptoethanol pure (Biopack, catalog number: 9545.05)

8. Diethyl pyrocarbonate (DEPC) (Sigma, catalog number: D5758)

9. Percoll (GE Healthcare, catalog number: 17-0891-01)

10. DEPC (diethylpyrocarbonate) (Sigma, catalog number: D5758)

11. SYBR^TM Safe DNA gel stain (Invitrogen, catalog number: S33102)

12. Agarose (Gene Biotech LE-Agarose 1200, catalog number: RU1010)

13. DEPC water (1 mL DEPC/L water; stirred overnight and autoclaved)

14. Phenol:chloroform:isoamyl alcohol 25:25:1 (Sigma, catalog number: P3803)

15. Chloroform (Biopack, catalog number: 1651.08)

16. Isopropanol (Mallinckrodt, catalog number: 3032-08)

Solutions

1. Buffer Tris HCl pH 8, 1 M (see Recipes)

2. EDTA pH 8, 0.5 M (see Recipes)

3. SDS 10% (see Recipes)

4. Ammonium acetate, 10 M (see Recipes)

5. Lithium chloride, 8 M (see Recipes)

6. Extraction buffer (see Recipes)

Recipes

1. Buffer Tris-HCl pH 8, 1 M

Weigh 12.11 g of Tris, add 75 mL of pure sterile water, adjust to pH 8 with HCl, and fill with pure sterile water to a final volume of 100 mL.

2. EDTA pH 8, 0.5 M

Weigh 18.6 g of EDTA, add 75 mL of pure sterile water, adjust to pH 8 using 10 M NaOH, and stir until EDTA is completely dissolved. Solubility improves while alkalinity increases; complete with pure sterile water to a 100 mL final volume.

3. SDS 10%

Dissolve 10 g of SDS in 100 mL of pure sterile water.

4. Ammonium acetate, 10 M

Dissolve 7.78 g of ammonium acetate in 10 mL of DEPC water.

5. Lithium chloride, 8 M

Dissolve 3.39 g of lithium chloride in 10 mL of DEPC water.

6. Extraction buffer (10 mL)

*Note: Weigh urea in a 50 mL Falcon tube and dissolve urea in half volume (5 mL for a final volume of 10 mL) of water. Vortex the solution to help it dissolve. The bottom of smaller tubes hinders the full solubility of urea. Then, add the rest of the components and complete to final volume. Keep at room temperature. Cold will precipitate SDS in the extraction buffer. No risk for later RNA extraction.

Laboratory supplies

1. Syringe, needles (13 × 0.3 mm) and pointy tips tweezers (Dumont, catalog number: 0103-2-PO)

2. Plastic grinding rod for Eppendorf tubes (SSIbio, catalog number: 1005-39)

Equipment

1. Magnifying glass (LABKLASS, model: HG468405)

2. Table centrifuge (Eppendorf, model: centrifuge 5418)

3. Vortex (Vornado^TM, Benchmark Scientific, catalog number/model: BV101-B)

4. Agarose gel electrophoresis equipment (Bio-Rad, model: PowerPac^TM Basic Power Supply, Horizontal Electrophoresis Systems)

5. NanoDrop^TM One/One (Thermo Fisher, catalog number/model: ND-ONE-W)

Procedure