DNA+Extraction

DNA Extraction Activity ................................................................................................................................................(Nod to James Dean)

Some basic, but cool, chemistry… DNA is the largest known molecule. A single unbroken strand can contain millions of atoms. When DNA is released from a cell it typically breaks up into short strand fragments. These smaller fragments have a slightly negative electric charge. Salt ions, common in many solutions, are attracted to the negative charges on the DNA fragments. By controlling the salt concentration of the solution, DNA can remain fragmented or become very “sticky” and form large globs of molecular material.

Releasing the DNA… The first step in obtaining DNA is to release the DNA from the cells of raw wheat germ. Detergents and soaps break down cell and nuclear membranes, releasing the DNA, and they also break up proteins that may harm the DNA. The released DNA dissolves in the water and will float upwards. The colored layer of liquid above the wheat germ will contain DNA and proteins. The DNA will then move up to a layer of alcohol carefully placed above the water. DNA doesn't stay dissolved in alcohol, but precipitates out and will appear as a white stringy mass.

Spooling the DNA on a stick… Alcohol allows DNA fragments to stick together, or precipitate, producing a blob of DNA that you can examine. The DNA precipitate can be captured, or spooled, onto a wooden stick by placing it in the DNA and turning it. If you want to save your DNA, you can transfer it to a small container filled with alcohol. (Source: [] )

Items needed Tall narrow container/flower vase (10mL graduated cylinder or test tube) Liquid dish soap or Woolite 90% Alcohol or higher Wheat Germ uncooked/not toasted Salt (extraction will work with out this.)

Procedure: Step 1: Fill your container 10 percent full of wheat germ. (1mL wheat germ) Also add 10 to 40 grains of table salt.

Step 2:, Add water to the wheat, germ filling until the container is 40 percent full. (4mL line)

Step 3 : Add about ½ percent of the container's volume of dish detergent the water. (small squirt or 0.3mL))

Step 4: Gently, gently, and even more gently stir the soap, water and wheat germ slowly. Let it set set 3 minutes, allowing the soap to work. (The soap will help break down the cellular walls and nuclear membranes of the wheat germ cells. This will free the DNA that is inside.)

Step 5: Fill your container up with Alcohol. (6mL) Put the alcohol in slowly by tipping your container slightly by running the alcohol down the side of the container.

Step 6: Watch the DNA begin to float up into the alcohol. Give it at least 10 minutes or more.

Step 7: Do the extraction more than once with different amounts of stirring to see its effect on the amount and form of DNA extracted.

(To improve the result you could try mixing the water and salt ahead of time. 2 grams salt to 90mL water. The alcohol could be chilled by placing the container holding it in an ice water bath.)

Lab Questions: 1. After step 6, what did you see in the alcohol and describe what it looked like? 2. What is another word or phrase for DNA? 3. What 4 nitrogenous bases are found in DNA? 4. What part of the cell did the DNA come from? 5. What usually keeps the DNA in its usual place in the cell? 6. What are cell membranes and nuclear membranes made of? 7. What does soap do to the stuff that membranes are made of? 8. Why do you think the soap helped to free the DNA? 9. DNA is less dense than water & alcohol. Why does the DNA move up into the alcohol? 10. We want the DNA to clump together, but like charges repel and DNA has a negative charge. Salt can provide a positive charge, so what is its purpose?

DNA and Membrane Facts: Copy as notes. 1. Living things are made of Nucleotides, Lipids, Proteins, and Carbohydrates. 2. DNA is a long, twisted, double chain of nucleotides that contains the genetic code. 3. Nucleotides are made a sugar, a phosphate and a base. (Complementary: A pairs with T and C pairs with G) 4. Membranes are made of lipids, such as the nuclear membrane. (Fats and oils are lipids) 5. Soaps cause lipids to break up in water, so soaps can be used to break up membranes. 6. Water attracts DNA, so it dissolves it, but alcohol repels DNA, so DNA forms a precipitate. 7. Opposite charges attract each other. In other words oppositely charges items neutralize each other when they come together.

Just read After the Exploration Expected Results ...A slimy white material will precipitate at the interface of the ethanol and filtrate layers. This material consists of clumped-together DNA strands and some protein. What's Going On? ...The procedure used in this activity has the same essential elements as more advanced laboratory DNA extraction procedures: mechanical and thermal disruption of cells, liberation of the DNA, and precipitation of the DNA. ...In this procedure, the wheat germ cell walls are broken down by the mechanical mashing and then the heating, and the detergent dissolves the lipids in the cell membranes and nuclear envelope (just like the detergent dissolves grease on your dishes). No longer confined inside nuclear membranes, the DNA--highly soluble in water because the phosphate group of each nucleotide carries a negative charge&emdash;goes into solution. However, the positively charged sodium ions from the salt in the extraction solution are attracted to the negatively charged phosphate groups on the DNA backbone, effectively neutralizing the DNA's electric charge. This neutralization allows the DNA molecules to aggregate with one another. When the ethanol is added, the DNA clumps together and precipitates at the water/ethanol interface because the DNA is not soluble in ethanol. ...Each glob of material in the precipitate will contain millions of DNA strands clumped together, along with some of the protein that is normally associated with DNA. (Since the DNA was not highly purified, some protein precipitates out with the DNA.) The yellowish water below the alcohol will contain dissolved proteins and some DNA that hasn't risen into the alcohol. (Source: [] )

DNA Extraction Lab Sheet:

Teacher Background:

DNA molecules are long, slender molecules that carry the heritable information of organisms on to future generations. Because of its microscopic size, it is impossible to see a DNA molecule with the naked eye. It would take about 300,000 DNA molecules side by side to make a bundle as thick as a human hair. When subjected to certain conditions, it is possible to collect “large” amounts of DNA to make it visible. buffer solution, made from soap, with the cell. The soap from the buffer solution disrupts the cell membrane’s phospholipid bilayer by reacting with the phosphate group of the phospholipid. This action releases the cell’s components into the buffer.

After you’ve mixed the buffer with the DNA source, you carefully overlay the buffer/DNA solution with isopropanol (rubbing alcohol); RNA and DNA precipitate in solutions containing high percentages of isopropanol or ethanol. Due to its size and abundance, chromosomal DNA forms viscous, clotted masses during such alcohol precipitation. You can use a plastic loop to mix the two liquids at their interface and to collect the DNA as it precipitates in the solution at the mixing zone.

Small fragments of DNA and degraded RNA usually contaminate the chromosomal DNA during extraction procedures. The alcohol also precipitates these fragments, but they have little tendency to spool on the loop because they are too short and form finer, more uniform precipitates. Therefore, you can view spooling as a method that partially purifies and concentrates high-molecular-weight DNA.

The purification of chromosomal DNA is frequently the first step in molecular-cloning experiments. Scientists can collect and redissolve the precipitate in a smaller volume. This is a convenient way to concentrate nucleic acids. Alcohol precipitations also remove small molecules, such as buffer salts, sugars, and amino acids, from nucleic acid precipitations because they remain in the solution.

http://www.scientificamerican.com/article/find-the-dna-in-a-banana-bring-science-home/ [] https://www.biotech.wisc.edu/docs/default-source/outreach-documents/dna-extraction-presentation2.pdf?sfvrsn=0

DNA is easily degraded by nucleases (enzymatic proteins), so DNA-protein interactions during DNA isolation must be limited. If the sample is kept on ice, protein activity drops to zero, so even if nucleases are present, they cannot act on and degrade the DNA. The ice bath is an indirect method for protecting the DNA until you can separate the proteins from the DNA.