Following is a simple technique for isolating and precipitating high molecular nuclear DNA from plant cells. A mortar and pestle are used to break cells open mechanically and to disrupt the plasma membrane. The plasma membrane is further degraded by the use of an extraction buffer containing a detergent that dissolves membranes. Their combined action produces a homogenate containing cell wall and plasma membrane fragments as well as intact nuclei, chloroplasts and mitochondria. Low-speed centrifugation is used to separate the nuclei from the smaller organelles.

OBJECT: To demonstrate how DNA is isolated from plant tissues.

MATERIALS: REAGENTS: Cauliflower homogenization solution, sodium citrate solution, sodium chloride
solution, Absolute ethanol.

           EQUIPMENT AND SUPPLIES: Centrifuge, Graduated cylinder, Funnel, Cheese-cloth, Centrifuge bottles, Centrifuge tubes, Chilled mortar and pestle, Glass rod, Razor blade, Fresh cauliflower head.

PROCEDURE:

1. Using a razor blade, remove 25g of the outer 2-3 mm of the cauliflower surface.

2. Place the tissue in a mortar and add 25 ml of sodium citrate solution. Grind the mixture until it becomes a smooth slurry.

3. Add 150 ml of cauliflower homogenization solution to the mortar. Continue grinding the mixture an additional 30 min.

4. Filter the homogenate through a funnel lined with two layers of cheese-cloth. Squeeze the cloth to recover any additional liquid. Transfer the liquid two centrifuge bottles.

5. Add 2 volumes of absolute ethanol to each bottle while stirring continuously. Balance the bottles and centrifuge them for 5 min at 200 x g at 4'c.

6. Pour off the supernatant, saving the pellet containing nuclei at the botton of the bottles.

7. Add 1.5 volumes of sodium chloride solution and stir the mixture.

8. Transfer the mixture to a pair of clean centrifuge tubes. Centrifuge them at 10,000 x g for 25 min at 20'c.

9. Save the supernatant in a clean beaker.Resuspend the pellet in 15 ml of sodium chloride solution.   Centrifuge the mixture at 10,000 x g for 25 min at 20'c.

10.Add the supernatant to the beaker containing supernatant from the previous centrifugation. Slowly add an equal volume of absolute ethanol while slowly stirring with a glass rod.

11. Fibrous DNA strands will collect on the rod. continue stirring until DNA no longer adheres to the rod.