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

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Isolation of DNA from Plant Material Practical

Studying the plant genome allows us to characterize and modify plant genes and metabolic pathways, as well as understanding the genetic variation in species. Transgenic plants are those that have been genetically modified using recombinant DNA technology. This can express a gene that is not native to the plant or to modify endogenous genes. The protein encoded by the gene will show a particular trait to that plant, for example, survival or growth. The RDT technology can be used to improve the nutritional content of the plant, as well as the production of some industrial products, such as monoclonal antibodies, vaccines, plastics and biofuels.

The method used for extracting DNA from plants is different from extracting genetic material from animal sources as the plant cells contain hard cellulose cell walls and large DNA molecules. Essentially, lysis of the cell wall and cellular membranes using mechanical or non-mechanical methods to allow access to genetic material, without its degradation. 

For this, usually, an initial grinding step is employed to break down cell wall material and allow access to DNA while cellular enzymes are inactivated. The main aim of this practical is to isolate DNA from available plant material and extract it in a pure form. 

Isolation of DNA Practical Aim

The aim of this practical is to isolate DNA from available plant specimens such as spinach, green pea, seeds papaya. 

DNA Isolation Practical from Plant Material Principle

Rupture or lysis of the cell wall and cellular membranes with the help of mechanical or non-mechanical methods. 

In the mechanical method, force is applied to the cell wall to open and spill the contents. In the non-mechanical method, enzymes or chemicals that specifically break down cell wall components are and with additional mechanical force. The advantage of mechanical disruption over the non-mechanical is that no chemicals are introduced in the cell solution that might interfere with the extracted cellular substance.

After lysis, small cracks are formed in the cell membrane for the accessibility of detergents. Chemical detergents break down the cell membranes due to their amphipathic. The DNA is then precipitated from the protein with ethanol. The clean DNA is now suspended in a 1xTE buffer or distilled H2O.

Materials:

  • Spinach/ papaya seeds / green pea

  • Cold ethanol

  • TE buffer

  • Distilled water

  • 100 ml detergent to 750ml of distilled water 

  • NaCl

  • Balance 

  • Centrifuge

  • Mortar and pestle

  • Gradual cylinder and beaker to keep the chemicals

  • Test tubes

  • Centrifuge tubes

  • Pipette

  • Enzymes (cellulose, ribonuclease etc) 

DNA Extraction from Plant Material Protocol

  1. The plant material is taken and then ground in a mortar using a pestle

  2. The plant material is treated with cell wall degrading enzymes for easy removal of the cell wall.

  3. The DNA extraction buffer is then added and a small amount of the solution is added in a test tube.

  4. In the same tube around 2 ml of ethanol is added (do not mix)

  5. DNA will appear as a clear white thread and it should be spooled using a Pasteur pipette.

  6. The DNA is transferred into a centrifuge tube and spun at 6000rmp

  7. The supernatant is gently removed and after keeping it for drying for some time, the DNA pellets are suspended in 0.5-1.5 ml of TE buffer.

[Image to be added Soon]

Results:  A cloudy precipitation of DNA is visible to the naked eye.

Isolation of DNA from Onion

An onion can also be used for this practical because it has a low starch content, which allows the DNA to be seen clearly. 

The salt used in the cell solution shields the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution.

Similar to other plant materials, the detergent causes the cell membrane to break down by dissolving proteins and lipids of the cell and disrupting the bonds that hold the cell membrane together. It then forms complexes with these biomolecules, causing them to precipitate out of solution.

Procedure

Add cold ethanol to the test tube to create an alcohol layer on top of about 1 cm. The alcohol can be added to the solution in three ways.

  1. Fill a Pasteur pipette with alcohol and then release it at the bottom of the test tube

  2. Put about 1 cm of alcohol into the bottom of a test tube and add the onion solution.

  3. Slowly pour the alcohol down the inside of the test tube with a Pasteur pipette.

DNA does not dissolve in alcohol. When alcohol is added to the cell solution, except the DNA, all the components of the mixture stay in solution. The DNA precipitates out into the alcohol layer.

Do not disturb the solution for 2-3 minutes. After that, the white DNA precipitate out into the alcohol layer. It can be spooled out using a glass rod and has a white mucus-like appearance.

FAQ (Frequently Asked Questions)

Q1: What are the Precautions That should be Taken During This Experiment?

Ans: 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 carrying 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.

Q2: What is Recombinant DNA Technology?

Ans: Recombinant DNA technology can be described as a technique in which the joining of DNA molecules from two different species that 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.

Q3:  What is the Role of the Following Substances in the Isolation of DNA from Plant Material Practical?

  1. Detergent

  2. Enzymes

  3. Chilled ethanol

  4. Salt

Ans: 

  1. Detergent: It dissolves the cell membrane.

  2. Enzymes: It digests the cell wall.

  3. Salt: Allows the DNA to precipitate

  4. Alcohol: Allows the DNA to precipitate on the alcohol layer.

Q4: Explain the Isolation of DNA from Papaya Seeds Practical.

Ans: Please have a look at the principle and procedure section of this article.

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