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.
Spinach/ papaya seeds / green pea
100 ml detergent to 750ml of distilled water
Mortar and pestle
Gradual cylinder and beaker to keep the chemicals
Enzymes (cellulose, ribonuclease etc)
DNA Extraction from Plant Material Protocol
The plant material is taken and then ground in a mortar using a pestle
The plant material is treated with cell wall degrading enzymes for easy removal of the cell wall.
The DNA extraction buffer is then added and a small amount of the solution is added in a test tube.
In the same tube around 2 ml of ethanol is added (do not mix)
DNA will appear as a clear white thread and it should be spooled using a Pasteur pipette.
The DNA is transferred into a centrifuge tube and spun at 6000rmp
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.
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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.
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.
Fill a Pasteur pipette with alcohol and then release it at the bottom of the test tube
Put about 1 cm of alcohol into the bottom of a test tube and add the onion solution.
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.