Class 12 Biology - Genetics and Evolution

Activities

Activity 2.1: DNA Isolation from Plant Material

Aim/Objective: To isolate DNA from a given plant sample (like onion or banana) and understand the biochemical role of each reagent used.

Materials Required:

Plant material (about 50g of Onion or Banana)
Lysis Buffer: (Prepared using 10ml liquid detergent + 5g NaCl + 90ml distilled water)
Chilled Ethanol (95%): Must be kept in a freezer for at least 24 hours.
Enzymes: Protease (Papain) or chilled Pineapple juice (optional but recommended)
Pestle and mortar, Beakers, Glass rod, Filter paper/Cheesecloth

Scientific Context: Why Chilled Ethanol? DNA is a polar molecule and is soluble in water but insoluble in ethanol. Adding ice-cold ethanol reduces the solubility further and helps DNA molecules aggregate and precipitate faster. The cold temperature also inhibits enzymes (DNases) that might otherwise degrade the DNA.

Procedure:

Maceration: Take 50g of plant tissue and crush it in a mortar with 20ml of Lysis Buffer.
Incubation: Keep the mixture in a water bath at 60°C for 15 minutes. This helps denature DNases.
Filtration: Filter the homogenate through a double-layered cheesecloth into a clean beaker.
Enzyme Treatment: Add 1ml of protease solution (or 5ml pineapple juice) and leave for 10 minutes. This removes histone proteins associated with DNA.
Precipitation: Tilt the beaker and slowly pour twice the volume of ice-cold ethanol along the side. Do not stir.
Spooling: Leave it undisturbed for 3-5 minutes. White, thread-like fibers will appear. Use a glass rod to gently "spool" the DNA.

Observation:

Fine, white, slimy, thread-like fibers precipitate out into the ethanol layer.

Explanation:

Detergent breaks the cell and nuclear membranes (lipid bilayer). Salt ( $NaCl$ ) provides $Na^+$ ions that neutralize the negative charge on the DNA backbone, allowing strands to come together. Ethanol precipitates the DNA out of the aqueous solution.

Conclusion:

Genomic DNA can be isolated by removing cellular debris, lipids, and proteins through sequential chemical treatment.

Activity 2.2: Observing Mitosis in Onion Root Tips

Aim/Objective: To study the different stages of mitosis in plant cells with high-precision staining.

Materials Required:

Onion root tips (collected between 8-10 AM)
Fixative (Farmer’s Fluid): Glacial Acetic Acid and Absolute Ethanol in 1:3 ratio.
Stain: 2% Acetocarmine.
1N HCl, 45% Acetic acid
Spirit lamp, Slides, Coverslips, Microscope

Glacial Acetic Acid The term "Glacial" refers to its pure form (99.8%+), which forms ice-like crystals at 16.6°C. It acts as a powerful fixative that penetrates tissues rapidly and prevents the shrinkage of chromosomes.

Procedure:

Fixation: Submerge fresh root tips in Aceto-alcohol (1:3) for 24 hours.
Hydrolysis: Place tips in 1N HCl at 60°C for 5 minutes.
Staining: Transfer to Acetocarmine for 15-20 minutes.
Squashing: Place tip on a slide, add a drop of 45% Acetic acid, heat gently for 2 seconds, and squash firmly under a coverslip using a thumb.

Observation:

Cells in Metaphase (chromosomes at equator) and Anaphase (chromatids at poles) are clearly visible.

Conclusion:

Precise fixation and hydrolysis are mandatory for clear chromosomal visualization.

Class 12 Biology - Genetics and Evolution

Activities

Activity 2.1: DNA Isolation from Plant Material

Aim/Objective: To isolate DNA from a given plant sample (like onion or banana) and understand the biochemical role of each reagent used.

Materials Required:

Plant material (about 50g of Onion or Banana)
Lysis Buffer: (Prepared using 10ml liquid detergent + 5g NaCl + 90ml distilled water)
Chilled Ethanol (95%): Must be kept in a freezer for at least 24 hours.
Enzymes: Protease (Papain) or chilled Pineapple juice (optional but recommended)
Pestle and mortar, Beakers, Glass rod, Filter paper/Cheesecloth

Procedure:

Maceration: Take 50g of plant tissue and crush it in a mortar with 20ml of Lysis Buffer.
Incubation: Keep the mixture in a water bath at 60°C for 15 minutes. This helps denature DNases.
Filtration: Filter the homogenate through a double-layered cheesecloth into a clean beaker.
Enzyme Treatment: Add 1ml of protease solution (or 5ml pineapple juice) and leave for 10 minutes. This removes histone proteins associated with DNA.
Precipitation: Tilt the beaker and slowly pour twice the volume of ice-cold ethanol along the side. Do not stir.
Spooling: Leave it undisturbed for 3-5 minutes. White, thread-like fibers will appear. Use a glass rod to gently "spool" the DNA.

Observation:

Fine, white, slimy, thread-like fibers precipitate out into the ethanol layer.

Explanation:

Detergent breaks the cell and nuclear membranes (lipid bilayer). Salt ( $NaCl$ ) provides $Na^+$ ions that neutralize the negative charge on the DNA backbone, allowing strands to come together. Ethanol precipitates the DNA out of the aqueous solution.

Conclusion:

Genomic DNA can be isolated by removing cellular debris, lipids, and proteins through sequential chemical treatment.

Activity 2.2: Observing Mitosis in Onion Root Tips

Aim/Objective: To study the different stages of mitosis in plant cells with high-precision staining.

Materials Required:

Onion root tips (collected between 8-10 AM)
Fixative (Farmer’s Fluid): Glacial Acetic Acid and Absolute Ethanol in 1:3 ratio.
Stain: 2% Acetocarmine.
1N HCl, 45% Acetic acid
Spirit lamp, Slides, Coverslips, Microscope

Procedure:

Fixation: Submerge fresh root tips in Aceto-alcohol (1:3) for 24 hours.
Hydrolysis: Place tips in 1N HCl at 60°C for 5 minutes.
Staining: Transfer to Acetocarmine for 15-20 minutes.
Squashing: Place tip on a slide, add a drop of 45% Acetic acid, heat gently for 2 seconds, and squash firmly under a coverslip using a thumb.

Observation:

Cells in Metaphase (chromosomes at equator) and Anaphase (chromatids at poles) are clearly visible.

Conclusion:

Precise fixation and hydrolysis are mandatory for clear chromosomal visualization.

Class 12 Biology - Genetics and Evolution

Class 12 Biology - Genetics and Evolution

Activities

Activity 2.1: DNA Isolation from Plant Material

Activity 2.2: Observing Mitosis in Onion Root Tips

On this page

Class 12 Biology - Genetics and Evolution

Class 12 Biology - Genetics and Evolution

Activities

Activity 2.1: DNA Isolation from Plant Material

Activity 2.2: Observing Mitosis in Onion Root Tips

On this page