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Isolate DNA From Available Plant Material Such As Green Pea, Spinach Seeds, Papaya

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Introduction

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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

Aim:

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


Procedure:

  • 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.


Observation

The DNA appears as white precipitates of fine thread on the spool.


Precautions

  • The leaf sample should weigh between 0.5 and 0.6 grams. 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.


Conclusion:

Isolating DNA from plant material is not just a scientific experiment; it's a journey of discovery. It allows us to appreciate the intricate dance of molecules within living cells and marvel at the power of DNA as the carrier of life's instructions. With each extraction, we gain a renewed sense of wonder for the diversity and complexity of the living world, and perhaps, even a glimpse into the fascinating story of our own existence.


So, the next time you bite into a juicy papaya or munch on a handful of green peas, remember the invisible blueprint hidden within each cell. With a little scientific curiosity and these simple techniques, you too can unlock the secrets of life, one DNA molecule at a time.

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FAQs on Isolate DNA From Available Plant Material Such As Green Pea, Spinach Seeds, Papaya

1. What is DNA isolation?

DNA isolation is the process of extracting deoxyribonucleic acid (DNA) from cells or tissues. DNA is the genetic material that carries hereditary information in all living organisms. Isolating DNA allows for its analysis and study in various fields, like genetics, medicine, and forensics.

2. Why isolate DNA from plants?

Plant DNA isolation is crucial for various research applications, including:


  • Plant breeding and genetic engineering: Studying plant genes helps develop improved varieties with desirable traits like disease resistance, higher yields, or better nutritional content.


  • Genetic diversity analysis: Investigating DNA variations within and between plant species provides insights into their evolution, adaptation, and conservation.


  • Plant forensics: DNA profiling can be used to identify plant species, track the origin of agricultural products, or investigate illegal logging or poaching.

3. Can I isolate DNA from plant materials at home?

Yes, isolating DNA from plant materials like green peas, spinach seeds, or papaya is a relatively simple process that can be done at home with readily available materials. However, it's important to note that homemade methods typically yield less purified DNA compared to professional laboratory techniques.

4. What materials do I need?

Here's a basic list of materials you'll need for plant DNA isolation:


  • Plant material: Green peas, spinach seeds, papaya flesh, etc.

  • Grinding tools: Mortar and pestle, blender, or zip-lock bag with rolling pin.

  • Extraction buffer: A solution containing salts, detergents, and enzymes to break down cell walls and membranes (homemade versions can be made with dish soap, NaCl, and Tris buffer).

  • Ethanol: For precipitating the extracted DNA

  • Test tubes or beakers: To hold the samples during the extraction process

  • Centrifuge (optional): Speeds up the separation of cellular components

5. What are the steps involved in isolating DNA from plants?

The basic steps involve:


  • Cell lysis: Breaking down the plant cells to release the DNA. This can be done by grinding the plant material with mortar and pestle, blending, or freezing and thawing.

  • DNA extraction: Using the extraction buffer to dissolve the DNA and separate it from other cellular components like proteins and cell wall debris.

  • DNA precipitation: Adding ethanol to the extracted solution causes the DNA to clump together and form a visible precipitate.

  • Purification (optional): Further steps can be used to purify the DNA and remove any remaining contaminants.

6. What are the Precautions That should be Taken During This Experiment?

For perfect results, all laboratory experiments must be carried out with some precautions in mind. The following are some precautionary steps that anyone should take whilst carry out this experiment.


  • All the glassware must be thoroughly cleaned and dried.

  • The ethanol used should be cold

  • If ordinary ethanol is used, the time duration for obtaining precipitated DNA may extend further.

  • The chemicals used for the experiments must be of standard quality.

7. What is Recombinant DNA Technology?

Recombinant DNA technology can be described as a technique in which the joining of DNA molecules from two different species are inserted into a host organism to produce new genetic combinations that are of value to science, agriculture and industry. rDNA molecules are produced using various laboratory techniques of RDT to bring together nuclear/genetic material from numerous sources, building genomic sequences that would not otherwise be found.

8. What is the Role of the Following Substances in the Isolation of DNA from Plant Material Practical?

1. Detergent

2. Enzymes

3. Alcohol

4. Salt

  1. Detergent: It dissolves the cell membrane.

  2. Enzymes: It digests the cell wall.

  3. Salt: Allows the DNA to precipitate

  4. Alcohol: This allows the DNA to precipitate on the alcohol layer.


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