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Isolate DNA from Plant Material
To isolate DNA from plant materials such as spinach, green peas, papaya and any other available plant material. Explore the procedures and process used in the experiment
CBSECBSE Class 12CBSE Class 12 Biology PracticalIsolate DNA from Available Plant Material Such as Spinach, Green Pea Seeds, Papaya
Isolate DNA from Available Plant Material Such as Spinach, Green Pea Seeds, Papaya
Isolate DNA from Available Plant Material Such as Spinach, Green Pea Seeds, Papaya Aim
To isolate DNA from plant materials such as spinach, green peas, papaya and any other available plant material.
Necessary Materials & Apparatus
Any available plant materials
Mortar and pestle Test tubes Beakers Ethanol Spool
Enzymes (Cellulase, ribonuclease, lipases, protease)
Take the available plant material and grind it in the mortar.
Treat the material with cellulase to break down the cell wall of the plant cells.
Next, treat it with protease to hydrolyze the peptide bonds of proteins in the plant material. In other words, the enzyme removes the histone proteins which are intertwined with the DNA.
Dissolve RNA with ribonuclease
Use lipase to dissolve lipids.
Add chilled ethanol to enable the precipitation of the DNA. It essentially increases DNA concentration.
Use spooling to extract the precipitated DNA. Spooling involves winding the fine threads of DNA on to a reel.
The DNA appears as white precipitates of fine thread on the spool.
Isolate DNA from available plant material such as spinach, green pea seeds, papaya, etc.
Isolate DNA from available plant material such as spinach green pea seeds papaya-etcExperiment 1: Study Of Pollen Germination on a slide
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the two types of nucleic acids found in living systems. DNA acts as the genetic material in most of the organisms. RNA, though, it also acts as a genetic material in some viruses, mostly functions as a messenger adapter, structural and in some cases as a catalytic molecule. All human knowledge, especially of natural sciences is directed to develop technologies for the comfort and well-being of human beings Biotechnology has emerged as an off shoot of modern biology in the twentieth century. The current break throughs in the field of biotechnology are production of genetically. modified organisms (plants, animals and micro-organisms) through recombinant DNA (r DNA) technology. Recombinant DNA technology (Genetic engineering, has allowed breeders to introduce foreign DNA in other organisms, including bacteria, yeasts, animals and plants). Such organisms are called Genetically Modified Organisms (GMOs). Thus, r DNA technology involves isolation of DNA from a variety of sources and formation of new combination of DNA.Objective. To isolate DNA from available plant material such as spinach leaves, green pea seeds, papaya etc.REQUIREMENTS
Plant material (such as spinach leaves, green pea seeds or green papaya), mortar and pestle, beakers, test tubes, enzymes (cellulase, protease, ribont1clease, lipases), ethanol, spool etc.PROCEDURE
Take/a small amount of plant material (such as spinach leaves/green pea seeds/green
pap/ya) and grind it in a mortar,
Break the cell wall and other envelope of plant cell, by treating the material by enzyme
Treat the material with enzyme protease to remove histone proteins which are intertwined
Dissolve the associated RNA with enzyme ribonuclease.
Also dissolve lipids with enzyme lipase.
Precipitate out the DNA by adding chilled ethanol.
Separate out the precipitated DNA as fine threads by spooling i.e., winding of the fine threads of DNA on real.
Theory How To Extract DNA From Anything Living
First, you need to find something that contains DNA. Since DNA is the blueprint for life, everything living contains DNA.
For this experiment, we like to use green split peas. But there are lots of other DNA sources too, such as:
Spinach Chicken liver Strawberries Broccoli
Certain sources of DNA should not be used, such as:
Your family pet, Fido the dog
Your little sister's big toe
Bugs you caught in the yard
Step 1: Blender Insanity!
Put in a blender:
1/2 cup of split peas (100ml)
1/8 teaspoon table salt (less than 1ml)
1 cup cold water (200ml)
Blend on high for 15 seconds.
The blender separates the pea cells from each other, so you now have a really thin pea-cell soup.
Step 2: Soapy Peas
Pour your thin pea-cell soup through a strainer into another container (like a measuring cup).
Add 2 tablespoons liquid detergent (about 30ml) and swirl to mix.
Let the mixture sit for 5-10 minutes.
Pour the mixture into test tubes or other small glass containers, each about 1/3 full.
Why am I adding detergent?
Step 3: Enzyme Power
Add a pinch of enzymes to each test tube and stir gently. Be careful! If you stir too hard, you'll break up the DNA, making it harder to see.
Use meat tenderizer for enzymes. If you can't find tenderizer, try using pineapple juice or contact lens cleaning solution.
Why Did I Add Meat Tenderizer?
Step 4: Alcohol Separation
Tilt your test tube and slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the pea mixture. Pour until you have about the same amount of alcohol in the tube as pea mixture.
Alcohol is less dense than water, so it floats on top. Look for clumps of white stringy stuff where the water and alcohol layers meet.
What is that Stringy Stuff?
DNA is a long, stringy molecule. The salt that you added in step one helps it stick together. So what you see are clumps of tangled DNA molecules!
DNA normally stays dissolved in water, but when salty DNA comes in contact with alcohol it becomes undissolved. This is called precipitation. The physical force of the DNA clumping together as it precipitates pulls more strands along with it as it rises into the alcohol.
You can use a wooden stick or a straw to collect the DNA. If you want to save your DNA, you can transfer it to a small container filled with alcohol.
You Have Just Completed DNA Extraction!
Now that you've successfully extracted DNA from one source, you're ready to experiment further. Try these ideas or some of your own:
Experiment with other DNA sources. Which source gives you the most DNA? How can you compare them?
Experiment with different soaps and detergents. Do powdered soaps work as well as liquid detergents? How about shampoo or body scrub?
Isolate DNA From Available Plant Material Such As Green Pea, Spinach Seeds, Papaya
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Isolate DNA From Available Plant Material Such As Green Pea, Spinach Seeds, Papaya
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Last updated date: 19th Jan 2023
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The extraction of deoxyribonucleic acid (DNA) from various sources is known as deoxyribonucleic acid (DNA) isolation. The methods used to isolate DNA vary depending on the sample's source, age, and size.
For genetic analysis, which is utilized for scientific, medicinal, or forensic objectives, DNA isolation is required. DNA is used in a variety of applications by scientists, including the introduction of DNA into cells, animals, and plants, as well as diagnostic purposes.
Isolation of DNA: Practical Procedure
DNA extraction from plant materials such as green pea seeds, spinach, papaya, and soon.
Requirements: Distilled water Spinach leaves Tris SDS EDTA Isopropanol Sodium acetate Chloroform Isoamyl alcohol
Phenol of the analytical grade
The cell wall and membranes are ruptured during homogenization, and the resulting product disperses in the buffer solution.
The cell extract is prepared by either manually rupturing the cells or chemically lysing them with enzymes.
In a mortar, grind around 0.5 gm of spinach leaf tissue with a pestle, then homogenise it with 2 ml of extraction buffer (100 mM tris, 20 mM EDTA, 0.5M NaCl, 7M Urea, 0.1 per cent - Mercaptoethanol, and 2% SDS make up the extraction buffer (pH 8.0).
After crushing, the tissue's long fibres are to be maintained, and the homogenate is to be transferred to a 2 mL microfuge tube.
The tubes are to be filled with an equal volume of phenol-chloroform (isoamyl alcohol 25: 24: 1) and gently shaken to blend properly.
Before beginning, the experiment tubes are to be centrifuged for 15 minutes at 15000 rpm at room temperature.
In a fresh tube, the upper aqueous phase is to be collected.
An equivalent volume of chloroform (isoamyl alcohol 24: 1) is stirred in.
The top aqueous phase is transferred to a new tube after centrifugation at room temperature for 10 minutes at 15000 rpm.
By adding 0.1 volume of 3M Sodium acetate pH 7.0 and 0.7 volume of isopropanol to the solution, the DNA is precipitated.
The tubes were centrifuged at 4'C for 15 minutes at 15000 rpm after 15 minutes of incubation at room temperature.
After that, the DNA pellet must be washed in 100 per cent ethanol and to be air-dried.
TE is to be used to dissolve the DNA (Tris-cl 10 nM, pH 8.0, EDTA 1mM).
RNA (10 mg/ml) is added to the DNA to get rid of RNA 5ul.
Ethanol precipitation in the presence of Na and at a temperature of - 20'C or below can be used to concentrate DNA samples.
The acquired DNA can be used for PCR, DNA fingerprinting, genome mapping, and recombinant DNA.
The leaf sample should weigh between 0.5 and 0.6 grammes. The leaf weight in the range of 0.5 gm to 1.0 gm and the DNA recovered to have a favourable correlation.
Standard pharmaceutical companies are to be taken into consideration for the chemicals required for DNA isolation.
To remove any dust particles, wash the plant material well with distilled water, wipe dry, and weigh it.
Recombinant DNA Technology Process
Recombinant DNA technology is a process that modifies the phenotypic of an organism (host) through the introduction and incorporation of a genetically modified vector into the host's genome.
As a result, the procedure comprises inserting a foreign DNA fragment into the genome that contains the target gene.
The introduced gene is referred to as a recombinant gene, and the procedure is referred to as recombinant DNA technology. It's not as simple as it sounds to embed a gene of interest into the host's genome.
Process of Recombinant DNA Technology
In recombinant DNA technology, the desired gene is chosen for injection into the host, followed by the perfect vector into which the gene must be incorporated, and so recombinant DNA is created.
The recombinant DNA must next be injected into the host. Finally, it must be preserved in the host and passed down to the offspring.
Fragmentation of DNA
Restriction endonucleases are used to cleave the DNA into pieces once it has been extracted and purified.
The restriction enzymes used in recombinant DNA technology are critical for detecting the precise position where the desired gene is inserted into the vector genome.
Restriction endonucleases cut DNA at certain locations and are sequence-specific, usually palindrome sequences.
They look at the length of the DNA and trim it at specific spots called restriction sites.
To obtain the complementary sticky ends, the appropriate genes and vectors are snipped by the same restriction enzymes.
As a result, ligases will have an easier time connecting the necessary gene to the vector.
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