A PLANT NUCLEAR DNA PREPARATION PROCEDURE

PMB Newsletter (1981) Vol. II
J. Bedbrook, CSIRO Division of Plant Industry, PO Box 1600, Canberra City ACT 2601, AUSTRALIA

This method can be used to prepare Petunia cv. Rose du Ciel nuclear DNA suitable for restriction enzyme analysis and cloning. Chloroplast DNA contamination will vary depending on the genotype. Greater chloroplast DNA contamination results when this method is used with the "Mitchell" line of Petunia.

1. Young leaves (20 g) from 1-3 month old Petunia plants are ground at 4 C with a minimal volume of 0.3 M sucrose, 5 mM MgCl2, 50 mM Tris HCl (pH 8) (HB) in a mortar and pestle. The slurry volume is brought to 100 ml with HB and filtered through 2 layers, then 4 layers of cheesecloth.
2. The filtrate is centrifuged at 1000 g for 5 min and the pellet resuspended in 100 ml of HB and repelleted at 1000 g for 5 min. This pellet is resuspended in 100 ml of HB containing 2% Triton X-100 incubated at 4 C for 10 min and centrifuged at 1000 g for 5 min. The pellet is washed with 100 ml of HB with Triton X100, recentrifuged, and the pellet cleaned of excess liquid with a paper towel.
3. The pellet is resuspended in 16 ml of 30 mM Tris-HCl (pH 8), 10 mM EDTA (RB), and transferred to a 100 ml flask. 2 ml of 10% (w/v) Sarkosyl is added along with 2 ml of 5 mg/ml pronase. The mixture is heated at 60 C for 5 min and incubated with gentle shaking at 37 C for 5-10 hrs. The solution is highly viscous and contains among other things, starch grains and nuclear debris.
4. After the incubation, measure the total volume of the lysate and bring it to a volume of 20 ml, Add 20 g of CsCl and dissolve completely over a period of about two hours at 4 C.
5. Bring the mixture to room temperature, add 2 ml ethidium bromide (10 mg/ml). mix in gently and thoroughly.
6. Dispense in centrifuge tubes and centrifuge at 35K rpm for 30 hrs at 15 C. Collect the DNA band under long wave uv light.
7. Recentrifuge the DNA and collect, extract the ethidium bromide with RB-saturated isoamyl alcohol and dialyze against 5 mM Tris-HCl (pH 8), 0.25 mM EDTA. Store at 4 C.

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 two 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 one layer of miracloth (Calbiochem) under four 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 (1-3X). 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 55-60 C for one 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 two layers of cheesecloth into a 38 ml quick-seal 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; ß-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