TO EXTRACT DNA FROM ONION – Bio Chemistry Practical

TO EXTRACT DNA FROM ONION – Bio-Chemistry Practical, To extract DNA from onion

Contents:-

AIM:

To extract DNA from onion

REQUIREMENT:

  • 250 ml Beaker
  • Sharp knife for cutting onion
  • large spoon for mixing
  • Blender
  • Thermometer
  • Strainer or funnel
  • Filter Paper
  • Hot water bath (60 degree C)
  • Ice water bath
  • Distilled water
  • Clear-colored shampoo or liquid detergent
  • large onion
  • Table salt, either iodized or non-iodized
  • (optional) Papain, a protease enzyme
  • Test tube
  • pipette
  • 95% ethanol

THEORY:

DNA, deoxyribonucleic acid, is the molecule of life. Every living organism has DNA in each cell of the organism and each molecule of DNA carries the blueprint for that organism. The DNA molecule is also responsible for heredity, passing on genetic information from parents to children. DNA molecules are large strands or chains of small molecules known as nucleic acids, which are localized in the nucleus of a cell. This process deals with breaking open plant cells(onion) and their nuclei to release the genomic DNA using a mechanical disruption and a protease to digest away the cell and nuclear walls. Once released, the genomic DNA is visualized by the addition of a precipitating solution (alcohol) and high salt, which causes the DNA to precipitate and become visible

PROCEDURE

  1. Set up hot water bath at 55-60 degree C and an ice water bath
  2. For each onion, make a solution consisting of one tablespoon (10 ml) of liquid dishwashing detergent or shampoo and 1/4 teaspoon (1.5 g) of table salt. Put in a 250 ml beaker. Add distilled water to make a final volume of 100 ml. Dissolve the salt by stirring slowly to avoid foaming.
  3. Coarsely chop one large onion with a knife . For best results, do not chop the onion too finely. It is better to have the pieces too large than too small.
  4. Mix chopped onion with the 100 ml of solution from step 2. The detergent dissolves the fatty molecules that hold the cell membranes
    together, which releases the DNA into the solution. The detergent, combined with the heat treatment used in step 5, causes lipids (fatty molecules) an proteins to precipitate out of the solution, leaving the DNA. The salt enables the DNA strands to come together.
  5. Put the beaker in a hot water bath at 55-60 degree C for 10-12 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down.
  6. Cool the mixture in an ice water bath for 5 minutes. During this time, press the chopped onion mixture against the side of the measuring cup with the back of the spoon. This step slows the breakdown of DNA.
  7. Filter the mixture through a filter paper using funnel. When pouring the mixture into the funnel, avoid letting foam get into the beaker. It can take more than an hour to recover most of the liquid.
  8. Transfer the solution to test tube. The test tube should contain about 1 teaspoon (4-5 ml) of solution. Stir the solution frequently when dispensing it into the tubes. The solution can be stored in a refrigerator for about a day before it is poured into the test tubes.
  9. Add 8-10 ml ice cold alcohol to the solution in test tube to create an alcohol layer on top . For best results, the alcohol should be as cold as possible. The alcohol is added to the solution by slowly pouring the alcohol down the inside of the test tube with a pipette or dropper. DNA is not soluble in alcohol. When alcohol is added to the mixture, all the components of the mixture, except for DNA, stay in solution while the DNA precipitates out into the alcohol layer.
  10. Let the solution sit for 2-3 minutes without disturbing it. It is important not to shake the test tube. You can watch the white DNA precipitate out into the alcohol layer. When good results are obtained, there will be enough DNA to spool on to a glass rod, a pipette that has been heated at the tip to form a hook, or similar device. DNA has the appearance of white mucus

RESULT:

The DNA was successfully extracted & isolated from the onion.

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