A Plant Nuclear DNA Preparation
- Grind in mortar and pestle or Waring blender with 5 to 7 volumes buffer
per 50 g tissue.. Use MCE at 350 l/L, and if necessary, with 5 mL
1 M DIECA/L.
- Squeeze through cheesecloth, 2 layers of Miracloth 10 minutes at 1000 g.
- Decant supernatant and centrifuge 10 minutes at 15900 g.
- Resuspend each pellet in a few drops of buffer G with paint brush; combine;
bring to about 10 mL/50 g, 15 mL/75 g.
- 10 min at 1000 g; pour off most; swirl pellet to remove fluffy layer; combine.
- Bring supernatant to 10 mM MgCl2 (100 mL 1M/10 mL). Bring to 20 g
DNAse/mL (100 mL 2 mg/mL/10 mL) 60 min at 4°C.
- Underlay shelf buffer, 20 mL/10–15 mL; always use 20 mL or more, 20
minutes at 12000 g.
- Resuspend in small volume shelf buffer with brush; bring to about
10 mL/50 to 100 g, 10 minutes at 15900 g.
- Resuspend pellets in NN (lysis) buffer (4–5 mL/50 to 75 g).
- Add SDS to 0.5% (250 mL of 10%/5 mL NN). Swirl thoroughly.
- Add proteinase K to 100 g/mL (20 mg/mL/5 mL NN). Swirl gently
60 minutes at 37°C.
- Add equal volume of 3:1 water-saturated phenol, chloroform-isoamyl
alcohol mixture. Emulsify 5 to 10 minutes at 7000 g.
- Collect supernatant; repeat 17 and 18: 3 total extractions.
- Final supernatant; add 0.1 volume 8 M Ammonium acetate; then add 2
volumes of absolute ethanol.
- 60 min, –80°C; 10 min at 8000–9000 g; drain; add equal volume 70%
ethanol; let sit 10 min; 10 min at 8000–9000 g; drain dry. Vacuum dry
pellet, 30 min. Two small corex tubes are better than one 130-mL Corex. Add 100 to 500 mL 0.1X NTE, 10 mL RNAse mixture. Typically use 500
mL per 50 g tissue.
- Hydrate 30 min, 37°C.
This method can be used to prepare petunias 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
- Young leaves (20 g) from 1- to 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.
- 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 BH with Tirton X100, recentrifuged, and the pellet cleaned
of excess liquid with a paper towel.
- 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.
- 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 2 hours at 4°C.
- Bring the mixture to room temperature, and add 2 mL ethidium bromide
(10 mg/mL). Mix in gently and thoroughly.
- Dispense in centrifuge tubes and centrifuge at 35K rpm for 30 hrs at 15°C.
Collect the DNA band under long-wave UV light.
- Recentrifuge the DNA and, extract the ethidium bromide with Rβ-saturated
isoamyl alcohol and dialyze against 5 mM Tris-HCl (pH 8), 0.25 mM
EDTA. Store at 4°C.