Extraction of Crude DNA from Plant and Animal Tissue IB Biology LAb Report

DNA is a universal code. Almost all organisms from bacteria up to the most complex organisms have the same genetic material. This includes plants, animals, humans, bacteria fungus... in this experiment we will be extracting DNA from animal tissue and plant tissue.

Part 1

Extraction of crude DNA from animal tissue:

Materials used:

• Fresh sheep liver
• Scalpel
• Blender
• Graduated cylinder
• Glass stirring rod
• Droppers 0.9% of NaCl solution
• Beakers
• Liquid soap
• 95% ethanol
• Cheesecloth

Procedure:

• Cut a piece of liver about 2 cm long. Put 30 ml of saline solution and the liver in a blender. Blend until the mixture is uniform.
• Fold two pieces of cheesecloth in half, and use it to strain the liver mixture into a beaker.
• Use a dropper to add approximately 30 ml of liquid soap to the mixture. Mix thoroughly with a glass stirring rod.
• Measure and record the volume of the soap-liver mixture in the beaker. Add a volume of ethanol twice your measured volume.
• Slowly stir the mixture with the glass stirring rod. A whitish substance will form where ethanol and liver mixture meet.

Data collection:

Qualitative data

Two phases are present in the ethanol-soap-liver mixture. An upper phase containing the ethanol is transparent and a lower phase which contains the liver is brownish. Slowly we can see a white substance diffusing into the ethanol layer. When we move the rod across this white substance, it starts to accumulate on the rod and clumps together.

Data processing and presentation:

The white substance diffusing into the ethanol layer is crude DNA. As we are stirring the rod DNA strands will accumulate and clump together on the rod.'

Conclusion and evaluation:

Crude DNA is insoluble soluble in ethanol that is why when the DNA diffuses into the upper layer, it is white and has and filament like form.

There are two layers, ethanol and liver mixture; the ethanol layer is the upper layer since it is less dense than the liver mixture.

The detergent, which is usually used to dissolve fats in a frying pan, in this experiment was used to dissolve the cell membrane. This is because the cell membrane is made of phospholipids (a type of fat). Fig 1

Fig 1

As the cell membranes dissolve, the cell contents flow out into the liquid where the cells were growing. Liver cell's DNA, their dissolved cell membranes and other contents of the cell become suspended and float in a cell "soap." However, DNA is soluble in water and cannot be seen in this soap , so we add some ethanol and the DNA will precipitate upward in the layer of alcohol.

One speculation about DNA is that it is a very long molecule, when we were stirring the rod and lifted it a bit from the surface of the mixture we could see long strands of white material (DNA) extending from the glass rod. When taking into consideration that this is a molecule, the distance it covers when it is extended is substantial. The Nacl dissolves in the solution giving Na+ ions, these ions then bond to the negative phosphate group of DNA which allows the molecules to clump together.

Part 2

Materials used:

• Material needed:
• I kiwi fruit
• 3% NaC1
• detergent ( washing up liquid)
• water bath set at 60 C
• crushed ice
• Scalpel
• filter paper
• funnels
• beakers/flasks
• test tubes and rack
• ice cold ethanol
• glass rod

Procedure:

• Chop kiwi fruit into small cubes, side about 0.5 cm.
• Add chopped kiwi fruit to 10 ml detergent+ 50 ml NaC1 solution, in flask or beaker.
• Hold flask/beaker in water bath at 60 C for exactly 15 minutes.
• Cool flask by holding it on ice.
• Filter through filter paper and discard kiwi fruit residue. DNA in now in solution in the filtrate.
• Transfer DNA solution to a test tube. Pour ice-cold ethanol carefully down the side of the tube on the surface to form an ethanol layer above the extract.
• Agitate the liquid by twisting a fine glass rod gently where the two liquids meet.

Data collection:

Qualitative data

We can observe two phases in the test tube. A lower green phase and an upper slightly transparent phase, the green phase containing kiwi "juice" and the transparent phase ice cold ethanol. We observe bubbles in the upper phase and a haziness forming. As we stir carefully the solution with a stirring rod, we can see that the haziness is increasing and that more bubbles are forming.

Data processing and presentation:

We inserted the kiw+NaCl+detergent into a flask and put the flask for 15 minutes in a 60C, and after we filtered the solution and added ice cold ethanol to it, two phases appeared, an upper transparent phase and a lower green phase. As we stirred using the glass rod the transparent phase started to change its color slightly and showed a type of haziness. Bubbles also started to appear in the upper transparent phase of the solution as the stirring continued.

Conclusion and evaluation:

The detergent is used to break down fatty (lipid) cell membrane to release DNA into solution by disrupting the polar interactions that hold the cell membrane together. The NaCl shields the negative phosphate ends of the DNA, allowing DNA to come together and precipitate out of cold alcohol solution, since the NaCl dissolves easily in water giving Na+ ions (which attach to the negative phosphate group). Heating was required since it is responsible for precipitating proteins and extraneous lipids out of solution leaving DNA behind. It also destroys enzymes that could break down DNA (DNAase). The cold ethanol allows only DNA to precipitate in it, leaving the other substances in the green phase of the solution. The ethanol had to be cold in order to slow the rate DNA break down.

Sources:

http://www.faculty.uaf.edu/ffdew2/geneticsWeb/lab/dnaLiver.pdf

http://www.omsi.edu/visit/tech/teachers/DNA.pdf

Advanced biology by Michael Kent