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DNA and Gentics the Extraction of DNA

AIM:  

To extract and observe DNA strands from onion tissue samples

HOW DOES IT WORK?                                        

Time allocation: 45 Min
Waiting time: 1-2 days

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, causes lipids (fatty molecules) and proteins to precipitate out of the solution, leaving the DNA. The salt enables the DNA strands to come together.

A Strawberry would be preferable as you can see the DNA separation more clearly due to the colour contrast. The salt shields are the negative phosphate ends of DNA, which allows the ends to come closer so the DNA can precipitate out of a cold alcohol solution. The detergent causes the cell membrane to break down by dissolving the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent then forms complexes with these lipids and proteins, causing them to precipitate out of solution.

One can also use Onion, Banana and Peas

APPARATUS:

  • 2x 1000ml Beaker
  • 5 ml Syringe
  • 2 x 250ml Beaker
  • Chattaway spatula
  • 2 x Test tubes 125 x 16mm
  • Thermometer 150mm -10/110C x 1.0div
  • Funnel 50mm
  • Retort stand
  • Retort rod
  • Beaker Clamp
  • Filter paper
  • Clear detergent soap 50ml
  • Sodium Chloride 5g
  • Test tube rack
  • Disposable dropping pipettes x 1
  • Ethanol Rectified 96%
  • Coarsely strawberry for best results or Onions etc.
  • Ice for cold water bath
  • Microscope
  • Microscope slide and cover glass

Supplied by the educator:

  • Knife
  • Strawberry
  • Ice for cold water bath

METHOD:

Preparation:
  1. Set up hot water bath by using a 1000ml beaker place it into the clamp once you set up the retort stand add (boiling water) measure up to 120˚C with a thermometer, and an ice water bath in the second 1000ml beaker by placing ice inside set this beaker aside.
  2. Make a solution consisting of 10 ml of clear liquid shampoo using the syringe (or a disposable pipette) and 1.5 g of Sodium Chloride to be measured on the scale.
  3. Placing the substances in a 250 ml beaker, slowly add water by tipping the contents of the beaker to the side so no foaming occurs until you reach the 100ml mark. Slowly agitate the solution to dissolve the sodium chloride salt still avoiding foaming to occur.
  4. Coarsely chop one large strawberry with a knife on the white tile and put into a 250ml beaker. For best results, do not chop the strawberry smaller than 5mm x 5mm.
  5. Cover chopped strawberry with the 100 ml of solution from step 2.
  6. Put the Beaker in a hot water bath at 120°C for 10-12 minutes, ensure the water in the water bath is higher than the level of the water inside the inner beaker with the strawberries. During this time, press the chopped strawberry mixture against the side of the beaker with the back of the spatula, an indication it is ready will be the softening of the fruit as you press it against the glass of the beaker. Do not keep the mixture in the hot water bath for more than 15 minutes because the DNA will begin to break down.
  7. Cool the mixture in an ice water bath for 5 minutes. During this time, press the chopped strawberry mixture against the side of the beaker with the back of the spoon. This step slows the breakdown of DNA.
  8. Filter the mixture through a Filter paper that is folded and placed into a funnel. When pouring the mixture into the strainer, avoid letting foam get into the measuring cup. It can take more than an hour to recover most of the liquid. The filtering can be done in a refrigerator overnight.
Experiment:
  1. Dispense the strawberry solution into test tubes you can do more than one. The test tube should be about 1/3 full. For most uniform results among test tubes, stir the solution frequently when dispensing it into the tubes. The solution can be stored in a refrigerator for a day before it is poured into the test tubes. When the solution is removed from the refrigerator, it should be gently mixed before the test tubes are filled.
  2. For best results, the alcohol should be as cold as possible. The alcohol can be added to the solution fill a disposable pipette with 5ml alcohol, put it to bottom of the test tube, and release the alcohol. 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.
  3. 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 Pasteur pipette that has been heated at the tip to form a hook, or similar device. DNA has the appearance of white mucus.
  4. Place the DNA on a Microscope slide and observe the DNA under the microscope.
  5. Make a scientific drawing of the DNA as observed under the microscope as per question 4 of the Student Practical questioner.

PRECAUTIONS:                                             

  • Be careful when cutting the onion into fine pieces as scalpel blade is sharp.
  • Watch the changes in temperature carefully as too high temperatures denature DNA.