Biology Experiment - Isolation Of DNA from Plant Materials
Introduction
Do you know humulin is the artificial insulin protein synthesized using rDNA technology? How is it synthesized? rDNA is the technology used to make insulin and other products. This technology also helps in the production of genetically modified organisms. rDNA technology involves the isolation of DNA and the formation of new DNA combinations. At present time all human knowledge is directed to the comfort and well-being of human life by developing various technologies. Biotechnology is also one such technology directed to comfort human life.
Table of Content
Aim
Apparatus Required
Theory
Procedure
Observations
Result
Precautions
Lab Manual Questions
Viva Based Questions
Practical Based Questions
Conclusion
Aim
The experiment aims to isolate DNA from plant materials such as papaya, green peas seeds, and spinach
Apparatus Required
The following apparatus is required for the experiment
Papaya, green pea, or any available plant material
Test tubes
Ethyl alcohol
Spool
Mortar and pestle
Enzymes( for digesting other cell material)
Beakers
Theory
DNA is the genetic material of all plant cells. It is found in the nucleus of all cells. Genetic engineering is the technique of altering the genetic material of organisms to bring changes. Now to modify genetic material we need to isolate it.
The following are the steps involved in the isolation of DNA from cells-
The cell is disassembled to release the DNA molecules as well as other macromolecules such as polysaccharides, proteins, RNA, and also lipids. This is done by treating the cells with different enzymes. Depending on the type of cell used for the isolation of genetic material different enzymes are used such as lysozyme used in bacterial cells, cellulase used in plant cells, and chitinase used in fungi.
Ribonuclease is the enzyme used to remove RNA from cells.
The proteins are removed using protease. These are the enzymes used to break protein.
Ethanol is added to precipitate the DNA.
The purified DNA can be collected.
Hence these are the steps used for the isolation of any genetic material from cells.
Procedure
First, take any available plant material and then grind it with the help of a mortar and pestle
After grinding mix it with cellulase enzyme, to break the cell wall of the cell.
After removing the cell wall, mix it with the protein-digesting enzyme protease.
Then RNA is digested by the ribonuclease enzyme
The lipid content of the cell is dissolved by using a lipase enzyme.
After removing or breaking all content of the cell, except DNA add chilled ethanol to facilitate the precipitation of DNA, it helps to increase the DNA concentration.
Precipitated DNA is extracted by the spooling process. Spooling process is defined as winding the fine threads of DNA on a reel
DNA Extraction
Observations
After extraction, DNA seems like a white precipitate of thread on the spool.
Results
The DNA is extracted and observed as a white precipitate.
Precautions
Following precautions must be taken while performing the experiment
The plant material should be washed with distilled or cleaned water to remove dust particles.
The enzymes and chemicals used in the experiment must be of standard quality
All instruments such as glassware must be cleaned and dried
Lab Manual Questions
1. Why is salt added to DNA?
Ans: Salt is always added to DNA because it facilitates DNA to precipitate when alcohol is added to the solution.
2. Why is a chilled condition required during the experiment?
Ans: The main reason for maintaining a chilled condition during the experiment is to protect DNA from cellular enzymes and also to increase the yield of DNA.
3. Why is the enzyme cellulase used for isolating genetic material from plants?
Ans: The cellulase enzyme is used for digesting the cell wall of plant cells.
4. What is biotechnology?
Ans: It is a branch of science that deals with the utilization of live organisms to make various products.
Viva Based Questions
1. What is recombinant DNA?
Ans: Recombinant DNA is the DNA formed by manipulating and Isolating DNA through various lab techniques.
2. What are genetically modified organisms?
Ans: Genetically modified organisms are made by altering the genetic material of organisms using genetic engineering techniques.
3. What is the role of detergent in the isolation of DNA?
Ans: Detergent is used to break the cell so that DNA can be released from the cell.
4. What are the roles of enzymes in the isolation of DNA?
Ans: Various enzymes are used to break the various content of the cell such as carbohydrate, protein, and RNA
5. What is biotechnology?
Ans: Biotechnology is the technique of utilizing live organisms, and their parts to make different products.
6. What types of nucleic acids are found in organisms?
Ans: Deoxyribonucleic acid and ribonucleic acid are found in organisms.
7. Describe the steps involved in the isolation of DNA.
Ans: The main steps of DNA isolation are - the creation of lysate, clearing of the lysate, binding, washing, and elution.
8. Why is DNA genetic material in most organisms?
Ans: DNA acts as genetic material in most organisms because it is chemically and structurally stable.
Practical Based Questions
Q1. Quantity of DNA in the given plant material can be estimated by which instrument?
spectrophotometer
Manometer
Thermometer
None of the above
Ans: (1) spectrometer
Q2. Which of the following chemicals is used for chilling ethanol?
Methanol
Ethanol
Acetaldehyde
None of the above
Ans: (2) Ethanol
Q3. Which of the following enzymes is used for the denaturation of protein while carrying out DNA isolation?
Chitinase
Lysozyme
Protease
All of the above
Ans: (3) protease
Q4. Which enzyme should be used for the isolation of DNA from fungus?
Protease
Amylase
Chitinase
All of the above
Ans: (3) chitinase
Q5. Which of the following methods is used for DNA isolation?
Phenol chloroform method
Silica gel membrane method
Salting out and proteinase k treatment method
All of the above
Ans: (4) All of the above
Q6. Which of the following is the step of DNA isolation?
Washing
Elution
Clearing of lysate
All of the above
Ans: (4) All of the above
Q7. Who discovered DNA isolation?
F. Miescher
Rosalind Franklin
Watson
None of the above
Ans: (1) F. Miescher
Q8. Double helix model of DNA was given by
Watson
Crick
Both of the above
None of the above
Ans: (3) Both of the above
Conclusion
In this article, we have learned an experiment on the isolation of DNA from available plant materials such as spinach, green seed pea, and papaya
We have also explained the DNA extraction diagram in this article
DNA can be isolated from any cell by following certain steps
After chilled ethanol is also added to facilitate the extraction process of DNA.
DNA is extracted in the form of white suspension thread on the glass rod.
FAQs on To Isolate DNA from Available Plant Materials Such As Spinach, Green Pea Seeds, and Papaya
1. What is the detailed procedure to isolate DNA from plant material like spinach or papaya, as expected in the CBSE Class 12 Biology practical exam for 2025-26?
To isolate DNA from a plant source, you should follow these key steps:
- Homogenisation: First, grind the chosen plant material (e.g., spinach leaves or papaya) with a mortar and pestle to create a fine paste. This breaks the cells mechanically.
- Lysis: Transfer the paste to a beaker and add a buffer solution containing enzymes like cellulase to dissolve the cell wall and a detergent to break down the cell and nuclear membranes, releasing the cell contents.
- Purification: Treat the solution with enzymes like protease to remove proteins (such as histones) and ribonuclease (RNase) to remove RNA.
- Precipitation: Gently pour chilled ethanol down the side of the beaker. Since DNA is insoluble in cold alcohol, it will precipitate out of the solution at the interface of the two liquids.
- Extraction (Spooling): The precipitated DNA will appear as fine, white threads. You can carefully wind these threads onto a sterile glass rod or spool to extract them from the solution.
2. What is the fundamental principle behind isolating DNA from a plant cell during the experiment?
The principle of DNA isolation is based on disrupting the cellular structure to release the DNA and then separating it from other macromolecules. The process involves:
- Breaking Barriers: The rigid plant cell wall (made of cellulose) and the cell and nuclear membranes are broken down using a combination of mechanical force (grinding) and enzymatic digestion (using cellulase and detergents).
- Removing Contaminants: Other macromolecules like proteins and RNA are enzymatically digested using protease and ribonuclease, respectively.
- Selective Precipitation: DNA is insoluble in chilled ethanol, while other components like sugars and salts remain dissolved. This property allows the DNA to be selectively precipitated and collected from the solution.
3. For the DNA isolation experiment, what is the specific reason for using chilled ethanol instead of room temperature ethanol?
Using chilled ethanol is a critical step for two important reasons:
- Decreased Solubility: DNA is much less soluble in cold alcohol compared to the aqueous buffer it is in. The cold temperature causes the DNA to precipitate out of the solution, making it visible as white threads and easy to collect.
- Enzyme Inhibition: The cold temperature inactivates any remaining cellular enzymes, particularly DNases, which could otherwise degrade the DNA you are trying to isolate. This helps in obtaining a higher yield of intact DNA.
4. Why are specific enzymes like cellulase and protease considered important for this experiment?
Each enzyme plays a highly specific and crucial role in purifying the DNA:
- Cellulase: This enzyme is essential for plant sources because it specifically targets and breaks down cellulose, the main component of the tough plant cell wall. Without it, the DNA would remain trapped inside the cell.
- Protease: Once the cell is broken open, the DNA is still tightly wound around proteins called histones. Protease digests these proteins, uncoiling the DNA and separating it from other cellular proteins, which prevents them from contaminating the final sample.
5. What are the key precautions a student must take to ensure a successful DNA isolation in the board practical exam?
To obtain a good yield of DNA and score well, you must observe the following precautions:
- Ensure all glassware, including the mortar, pestle, and beakers, is thoroughly clean and preferably sterilised to avoid contamination.
- Maintain a chilled environment for all solutions, especially the ethanol, to maximise DNA precipitation and prevent degradation.
- When adding the chilled ethanol, pour it slowly and gently along the inner side of the test tube or beaker to form a distinct layer over the cell lysate. Avoid mixing the layers vigorously.
- Handle the fine DNA threads gently during spooling to prevent them from breaking.
6. How would the initial step of DNA isolation differ if you were using a bacterial or fungal cell instead of a plant cell?
The primary difference lies in the enzyme used to break down the cell wall, as its composition varies between organisms. The rest of the procedure remains largely the same.
- For a Bacterial Cell: You would use the enzyme lysozyme, which specifically breaks down the peptidoglycan layer of the bacterial cell wall.
- For a Fungal Cell: You would use the enzyme chitinase to digest the cell wall, which is primarily made of chitin.
- For a Plant Cell: As in this experiment, cellulase is used to digest the cellulose cell wall.
7. After isolating the DNA as white threads, what does this observation signify and how can its purity be checked?
The observation of a mass of fine, white, thread-like precipitate at the alcohol-lysate interface is a positive result, indicating that DNA has precipitated from the solution. To further analyse it:
- Visual Confirmation: The thread-like appearance itself is a strong indicator of long-chain polymeric DNA.
- Purity Check: The purity of the extracted DNA is professionally measured using a spectrophotometer. The ratio of absorbance at 260 nm to 280 nm (A260/A280) is calculated. A ratio of approximately 1.8 is considered indicative of a pure DNA sample, free from major protein contamination.
