Joseph Chalhoub Biol 275Lab Inst.

: Nedal Taha

DNA Isolation from Onion.
I. Introduction:

Scientists are involved with research in finding out as much as possible about the DNA in plants and animals. Although DNA was discovered in the 1950 s, there still remains a lot to be known about it, especially how it regulates the workings of the body and how it is used to determine the corporeal traits that we all have. We should always remember that DNA is just a chemical named deoxyribonucleic acid. Because it is a chemical, we can do reactions with it just like we can work with any other chemical. In this lab, we will use the chemical properties of DNA to extract it from the cells of onions.

Nucleic acid isolation usually consists of three steps : (1) disruption of cell membranes with the cell wall in some organisms and the membrane of the subcellular nucleus to release the nucleic acids,(2) dissociation from nucleoproteins and denaturation of protein (3) separation of DNA from other soluble cellular components.
The purpose of this laboratory is to give you firsthand experience with DNA by isolating it from plant tissue. You will start with whole onions and end with a relatively pure preparation of DNA, containing literally millions of genes. Once isolated, the DNA can be stored in alcohol or dried out. It will actually be possible for you to hold in your hands the key to an organism's development and structure. II. Objectives

1. To become familiar with the physical properties of DNA by isolating it from living tissue

because inside the cell is where the DNA is.10% mL SDS IV. 5) Heat the solution. 3) Transfer the blended onion into a 250mL. the saltiness and acidity (pH) of the solution is very close to that in living things. It will be important that these cell walls break down in this lab. Secondly. Because detergent is used to break apart greasy particles in your clothes. homogenate in a blender for about 40seconds. as a result. 2) Adding distilled water. Use a mortar and pestle to mash the pieces of onion into a pulpy sludge. To learn the purpose of each step in the isolation procedure as it relates to the physical and biochemical characteristics of the genetic material III. the detergent is added to help break down cell walls in the onion cells.2. Cell walls in living things are made of long polar molecules with a greasy end and a charged end. Materials: y y y y y y y y Fresh onions Detergent Distilled water Ethanol (95%) Graduated Cylinders (10mL and 100mL) Knife Beaker Glass stirring rod Solutions: -Lysing buffer1: 1M TRIS 5M EDTA 5M NaCl Distilled water . 4) Add 1mL of 10% SDS and stir. Half an onion should be plenty for this lab. 1 This buffer solution is used in this lab for several reasons. First of all. it will also work to tear apart the greasy molecules in cell walls. 7) Transfer to a test tube using a 50mL pipette. and then add ethanol (ice cold) slowly pipetting down the side of the tube. Procedure: 1) Dice an onion with a knife. . the DNA will like to dissolve into this solution. using a funnel. 6) Let the mixture cool for 5 minutes.

V. Why we need to mash the onion: What we want to do by mashing the onion is to either break the cell walls (releasing the DNA into the juice) or at the very least expose the cell walls so the detergent can break them down. Why we need to chill the buffer solution: As important as DNA molecules are to life. Conclusion: DNA molecules are highly complicated and essentials compounds in the organism. Chemical reactions always take place slower in cold solutions than in warm ones. and their method of isolation from the rest of the cell is made of indispensable yet simple stages of breaking the cell membranes. We can actually see the basic source of life after this experiment. To slow down the rate at which the DNA breaks up. VII. because there is a lot less energy around to make the reaction take place. we cool down the buffer solution to near freezing. Observation: A clear layer of ethanol should form on top of the onion filtrate. which minimizes change in the acidity of a solution when an acid or base is added to the solution thus maintaining the pH almost constant. and break apart easily when removed from cells. they are still extremely fragile. . Questions: 1) Buffer: a mixture of an acid and its conjugate base. VI. Precipitate: a solid separated from a solution in which the solid is not soluble. (DNA is not soluble in ice cold ethanol) therefore DNA precipitates out of the solution becoming visible as white strings in the ethanol layer. seperationg the DNA from proteins and RNAs and finally precipitating the DNA molecules.

Filter: A porous material through which a liquid or gas is passed in order to separate the fluid from suspended particulate matter. SDS: anionic detergent. Sodium cations are essential in preserving this structure because hydrogen bonds are lost in a medium with low ionic forces. . pineapples. releases the plasmid DNA and sheared cellular DNA. 3) Na + Negatively charged DNA DNA has a double helical structure maintained by hydrogen bonds. Emulsify: to form emulsions (small globules of one liquid in another liquid with which the first will not mix) 2) The lysing buffer is used to maintain pH and other roles: EDTA: destabilizes the cell membrane. disrupts ionic interaction between proteins. denatures the DNA of the cell. apples y Some fruits and vegetables contain polysaccarides which make purification of the DNA more challenging. prevents precipitation of DNA. 4) Other vegetables and fruits that can be used for this lab are: Tomatoes. bananas. inhibits DNAses and binds divalent metal ions that could form salt with anionic phosphate group of DNA. cherries. NaCl: loosens the cell wall for increase solubility and stability of DNA.

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