Lab 2 DNA Extraction PDF

DNA Extract ion Lab # 2 Theoretical background  The Cell All living things are made of cells. Cells are the basic unit of life and make up all plants, animals and bacteria. In plants and animals, cells often work together to form tissues; groups of these tissues are called organs....

DNA Extract ion Lab # 2 Theoretical background  The Cell All living things are made of cells. Cells are the basic unit of life and make up all plants, animals and bacteria. In plants and animals, cells often work together to form tissues; groups of these tissues are called organs. For example, heart cells make up heart tissue, which in turn makes up the organ called the heart. The cells in your heart work together to push red blood cells through your body. Red blood cells carry oxygen to all parts of your body and oxygen is used to produce energy so your body can survive.  The Nucleus Inside cells are smaller structures called organelles. These tiny structures act like factories that help the cell perform certain tasks such as general repairs, removing waste, and reproduction. The three main parts of the cell are the nucleus, which holds DNA, the cell membrane, which surrounds and protects the cell, and the cytoplasm, which is the jelly-like part of the cell between the membrane and the nucleus. All of the smaller organelles, such as mitochondria, are found in the cytoplasm. What is DNA? DNA is present in all living things from bacteria to plants to animals. DNA contains the genetic code or commands that direct cellular activities and ultimately, the tissues, the organ and the body. In animals, it is found in almost all cell types except for usually in red blood cells. DNA is made of two spiral strands that wind around each other like a twisted ladder. Always packaged with Histone proteins. DNA is Deoxyribonucleic acid. It contains a chain of Nucleotides (each nucleotide contains a base – a sugar and a phosphate), The sugar is Deoxyribose. The bases are adenine (A), thymine (T), cytosine (C), and guanine (G). The bases pair together by hydrogen bonds: adenine with thymine and cytosine with guanine. These base pairs make up the rungs of the ladder. The different nucleotides are like a four-letter alphabet and can spell out different codes. A gene is a long series of codes that contains instructions for the cell to make a particular protein. Practical Experiment DNA extraction DNA is the largest known molecule. A single unbroken strand contains millions of atoms. After adding the detergent The DNA is released from a cell and breaks up into tiny fragments. These tiny fragments have a negative electric charge. Salt ions, common in many solutions, are attracted to the negative charges on the DNA fragments and this allows the many separated DNA fragment to come together. By controlling the salt concentration of the solution containing the DNA fragments, DNA can remain fragmented or become very “sticky” and form large globs of molecular material. Objective To extract DNA from plant cells To understand the steps needed to Extract DNA from plant cells To visually observe the DNA structure To understand the general structure of DNA Steps in DNA Extraction The process of isolating DNA requires that it be released from a cell whether it is a plant (which has extra protection with a cell wall), animal, fungi, or bacterium. Detergents or soaps breakdown cell membranes and proteins so that the DNA can be released. Salt will neutralize the negatively charged DNA and allow it to come together like glob of molecules Once the DNA fragments are released into solution, a small layer of alcohol is added to the top of the solution containing the cellular fragments. During alcohol precipitation, DNA becomes insoluble and aggregates into visible clumps. This process happens because DNA is insoluble in alcohol and sticks together in a visible mass when gently pulled. The DNA can be spooled together by using ice-cold alcohol. The DNA will collect at the interface between the alcohol and the cell solution. The DNA can then be captured, or spooled, onto a wooden stick or glass rod. The alcohol allows the DNA fragments to stick together once again, and you have a blob of DNA to examine. Although this method is effective at isolating DNA, the DNA is by no means pure. Other materials like protein and cell fragments are carried along. Procedure Crushing 1. Add kiwi/strawberry fruit into extraction solution (Salt and detergent) in the zipper bag. Close bag and squeeze out air. Add lysis buffer (salt and detergent) 2. Crush the kiwi/strawberry thoroughly for 5 minutes. CAREFUL don’t break the bag! 3. Tape the cheese cloth over the beakers. Filter the fruit mixture through the cheese cloth. Let the solution drain 5 minutes. Filter 4. Using the large transfer pipettes, aliquot approximately 2 ml of the kiwi/strawberry fruit solution into a test tube. 5. Add approximately 2 ml of ice-cold ethanol to each tube by dropping it slowly down the side of the test tube, allowing it to rest on top of the kiwi/strawberry fruit mixture. Do not agitate the solution. Add ice cold Ethanol 6. Let the solution sit for two minutes without disturbing it. The DNA will appear as transparent, slimy, white mucus which can be spooled up with the wood applicator stick. Observe DNA Watch the DNA extraction video Procedure Questions 1. Why do we “crush” the kiwi/strawberry fruit? 2. Why do we use shampoo? 3. What does the salt do? 4. Why do we need to cool the mixture? 5. What does the cold ethanol do? 6. Why can’t we use room temperature ethanol? Why do we “crush” the kiwi/strawberry fruit? 1.Crushing the kiwi/strawberry fruit physically breaks apart the cell walls. 2.To create a more malleable material, almost to the consistency of a liquid. Why do we use detergent? 1. After the cell walls have been disrupted during mechanical mashing of the fruit, the detergent in the shampoo disrupts the cell and nuclear membranes of each cell to release the DNA. It does this by dissolving lipids and proteins that hold the membranes together. 2. When soap comes close to grease, their similar structures cause them to combine, forming a greasy soapy ball. 3. A cell's membranes have two layers of lipid (fat) molecules with proteins going through them. 4. When detergent comes close to the cell, it captures the lipids and proteins. What does the salt do? 1. The salt neutralizes the negative charges on the DNA and thus enables the DNA strands to stick together. 2. It also causes proteins and carbohydrates to precipitate. Why do we need to cool the mixture? 1. DNAses or restriction enzymes that destroy DNA are present in the cell’s cytoplasm. 2. They are there to protect the cell from invasion by viruses. 3. Once the nuclear membrane is destroyed by the soap, the DNA is now susceptible to the DNAses and will quickly be degraded. 4. However, these enzymes are temperature sensitive and cooling the solution slows down the process of degradation. What does the ethanol do? 1. Everything except the DNA will dissolve in Cold Ethanol ethanol. 2. The ethanol pulls water from the DNA molecule so that it then collapses in on itself and precipitates. 3. The DNA will become visible as white mucous strands that can be spooled with the wooden applicator stick. Why do we use alcohol? Alcohol is less dense than water, so it floats on top. Look for clumps of white stringy stuff where the water and alcohol layers meet. DNA is a long, stringy molecule. The salt that you added in step one helps it stick together, so what you see are clumps of tangled DNA molecules! DNA normally stays dissolved in water, but when salty DNA comes in contact with alcohol it becomes undissolved. This is called precipitation. The physical force of the DNA clumping together as it precipitates pulls more strands along with it as it rises into the alcohol. You can use a wooden stick or a straw to collect the Why can’t we use room temperature ethanol? The colder the ethanol is the greater the amount of DNA that is precipitated. Using room temperature ethanol allows more DNA to be susceptible to the action of DNAse which will be active at room temperature and therefore, you observe less DNA.  Do the lab assignment Prepare for the lab quiz