Plant DNA Extraction

A thaliana has a very small haploid genome, and this makes obtaining DNA somewhat difficult. The most notable problem is that DNA is usually contaminated with polysaccharides, which inhibit restriction enzymes, as well as other DNA-modifying enzymes. This problem is most easily solved by using young plants that have not accumulated as much polysaccharide as older plants. The best results are obtained with plants that are 2 to 3 weeks postgerminated.

1. Harvest plants using forceps—carefully remove any adhering soil by hand.
2. Grind up the following in a mortar and pestle until no large pieces of tissue remain:
   - 0.5–1.5 g plants
   - 0.5 g of glass beads (75-150 µm) per gram of plants
   - 3 mL proteinase K buffer (0.2 M Tris (pH 8.0), 0.1 M EDTA, 1% Sarkosyl, 100 g/mL proteinase K)
3. Pour into a 10 mL test tube. Incubate at 45–50°C for 1 hr.
4. Spin 10 minutes at top speed in table top centrifuge (~3000 rpm)
5. Decant supernatant to a fresh tube. Adjust volume to 3 mL with proteinase K buffer (with or without proteinase K).
6. Add 6 mL 100% ethanol at room temperature. Invert to mix.
7. Spin 10K rpm for 15 min in SS34 rotor. Discard supernatant.
8. Resuspend pellet in 3 mL Tris-Cl (pH 8.0), 1 mM EDTA (TE). Vortex to resuspend.
9. Extract with phenol, phenol:chloroform, chloroform.
10. Add 6 mL 100% ethanol. Invert to mix.
11. Spin 10K rpm for 15 min in SS34 rotor. Discard supernatant. Air dry pellet briefly.
12. Resuspend in 4 mL TE. Vortex to resuspend.
13. Add 4.5 g CsCl, 10 mg/mL ethidium bromide and mix.
14. Spin 53K rpm 16–20 hrs VTi65 20°C.

This protocol has been optimized for yield at the expense of high molecular weight DNA. The nuclear DNA can be separated from plastid DNA by running the gradients with Hoest dye, rather than ethidium bromide.

Plant DNA Isolation

1. Prepare 5–20 g of clean, frozen, young leaves taken from plants grown under controlled conditions and exposed to darkness for 2 days prior to isolation. Remove mid-ribs.
2. Grind leaves in stainless steel blender containing 150–200 mL of ice-cold H buffer at maximum speed for 1 min.
3. Pour the homogenate in a 250-mL centrifuge bottle (on ice) while filtering through 1 layer of miracloth (Calbiochem) under 4 layers of cheesecloth (all previously wetted with 10 mL clod H buffer).
4. Centrifuge at 2000 g, 4°C, 20 min.
5. Discard the green supernatant and resuspend the pellet in 40 mL ice cold HT buffer.
6. Transfer to a 50-mL teflon tube (Oakridge OK) and centrifuge at 2000 g, 4°C, 10 min. Repeat until pellet of nuclei becomes greyish-white (1 to 3X). If anthocyanins are present in the plant, the pellet will be reddish-brown.
7. Resuspend the pellet thoroughly in 12 mL of HT buffer, then add 12 mL of lysis buffer.
8. Immediately, add 23.28 g of powdered CsCl and incubate the tubes at 55°C–60°C for 1 hour with occasional inversion.
9. After solubilization of the CsCl, centrifuge the tubes at 28000 g, 15°C, 30 min.
10. Filter the supernatant through 2 layers of cheesecloth into a 38-mL quickseal tube containing 1.47 mL EtBr solution using a 50-mL syringe and a 16-G needle as a funnel. Complete volume with CsCl solution.
11. DNA is recovered after centrifugation using standard procedures.
    1X TE: 10 mM Tris-HCl (pH 8), 1 mM EDTA
    1X H: 10X H, 400 mL; sucrose, 684 g; β-mercaptoethanol, 8 mL; water to 4000 mL [pH 9.5]
    Lysis buffer: Na-sarcosine, 4 g; Tris base, 2.42 g; Na2EDTA, 2.98 g; water to 200 mL [pH 9.5]
    10X H: spermidine, 20.35 g; spermine, 27.8 g; Na4EDTA 83.24 g; Tris base, 24.2 g; KCl, 119.2 g; water to 1900 mL [pH 9.5]; add Phenyl methyl sulfonyl fluoride (PMSF) solution [7 g in 100 mL of 95% ethanol]
    1X HT: 1X H, 1000 mL; Triton X100, 5 mL
    CsCl solution: CsCl, 97 g; 1X TE, 100 mL
    EtBr solution: EtBr, 1 g; water, 100 mL.