Experiment 5: Isolation of DNA from Available Plant Material

AIM

To isolate DNA from available plant material like pea seed, spinach, onion, etc.

PRINCIPLE

Plant cells contain DNA within nucleus and organelles protected by cell wall, cell membrane and histone proteins. Isolation requires breaking these barriers and removing associated proteins, RNA and lipids to obtain pure DNA.

Cellulase digests cell wall, protease removes histone proteins bound to DNA, ribonuclease degrades RNA and lipase dissolves lipids. Ice-cold ethanol precipitates DNA because DNA is insoluble in alcohol and appears as white threads.

Spooling with glass rod allows collection of long DNA fibres. The method demonstrates that DNA is present in all plant cells and can be extracted from easily available materials like onion, spinach and pea seeds.

REQUIREMENTS

• Plant material (like pea-seed, spinach leaves, onion or cauliflower shavings)
• Mortar and pestle
• Beakers
• Test tubes including boiling test tube
• Muslin cloth
• Enzymes like cellulase, protease, lipase and ribonuclease
• Ethanol - ice-cold 95% ethanol
• Spool
• Glass rod
• Distilled water

PROCEDURE

1. Take small amount of plant material (onion or cauliflower shavings) and grind it in a mortar with the help of pestle. Alternatively, the plant material can be homogenised in a blender and can be filtered easily through the muslin cloth.
2. Now, pour the filtrate into a boiling test tube.
3. Break the cell wall and envelope of plant cell by treating the material with the enzyme cellulase.
4. Now, the same material is treated with enzyme protease to remove histone proteins.
5. Now, add ribonuclease in it to dissolve the associated RNA followed by addition of lipase to dissolve the lipids present in the sample.
6. Gradually pour twice the volume of ice-cold 95% ethanol into the test tube. This will allow DNA to precipitate out.
7. The precipitated DNA can be spooled with the help of glass rod. It appears as the winding of the fine threads of DNA.

OBSERVATION

Shiny white DNA can be seen at the junction of solution and ethanol. This DNA represents all the DNA found in plant cells.

The chromosomes were broken in the process and the DNA gets precipitated due to the chemical treatment.

Illustrative observations for different plant materials (for reference):

RESULT

DNA appears as white precipitate of very fine threads on the spool. Spooling confirms successful isolation of DNA from the given plant material.

CONCLUSION

DNA can be isolated from easily available plant materials using enzymatic treatment and ethanol precipitation. Presence of white thread-like precipitate confirms that DNA is a stable macromolecule present in all plant cells.

PRECAUTIONS

• Fresh plant materials should be used for extraction of DNA.
• The glass wares must be thoroughly cleaned and dried before the experiment starts.
• The enzymes and chemicals used for the experiment should be of standard quality.
• Always use distilled water to make the solutions.
• Chemicals and solutions should be prepared carefully in order to avoid any wastage.