OBJECTIVE To extract and isolate DNA from a banana by following common DNA spooling techniques

INTRODUCTION DNA isolation is a process of purification of DNA from sample using a combination of physical and chemical methods. Currently it is a routine procedure in molecular biology or forensic analyses. DNA molecules are long, slender molecules that carry the heritable information of organisms on to future generations. Because of their size, it is impossible to see a single 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. This process of collecting DNA is referred to as spooling. During spooling, a buffer solution made from soap and salt (NaCl) is mixed with the cells. The soap from the buffer solution disrupts the cell membranes phospholipid bilayer by reacting with the phosphate group of the phopholipid. This releases the cellular components into the buffer. Once the components are released, the NaCl in the buffer binds to several of the negatively charged phosphate groups of the DNAs sugar-phosphate backbone, shielding some of the negative charge of the DNA. Because some of the negative charges are shielded, the DNA molecules can loosely bind together. After mixing the buffer with the DNA source, the buffer/DNA solution is carefully overlaid with isopropanol. In the presence of salt, RNA and DNA precipitate from solutions containing high percentages of ethanol or isopropanol. Due to its size and abundance, chromosomal DNA forms viscous, clotted masses during alcohol precipitation. A plastic loop is used to mix the two liquids at their interface and collects the DNA as it precipitates from solution at the mixing zone. Small fragments of DNA and degraded RNA usually contaminate the chromosomal DNA during extraction procedures. They are also precipitated by the alcohol, but have little tendency to spool on the loop because they are too short and form finer, more uniform precipitates. Spooling can consequently be viewed 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. The precipitate can be collected and redissolved 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 since they remain in solution.

PROCEDURE 100 ml beaker was used to prepare the buffer using 60 ml of tap water, 0.75 g (1/8 tsp) of sodium chloride, 2.5 g (1/2 tsp) of sodium bicarbonate and 2.5 ml of detergent (dish wash). The solution was stirred well and chill buffer in freezer or ice for 15 minutes. A whole banana fruit was cut into half. By using spatula and mortar, the banana has smashed until pureed. Half spatula of the banana puree was transferred into a clean 50 ml beaker. 10 ml of chilled buffer solutions was added and stirred vigorously for at least 2 for minutes. Filter paper in filter funnel was used and the banana was poured in the buffer mixture into the filter. The filtered liquid was collected in a test tube. 10 ml of ice cold isopropanol was added slowly into the mixture in the test tube The solution sits for 2-3 minutes without disturbance. Shaking was not recommended for the solution. The precipitation of white DNA into the isopropanol layer was observed.

RESULT

DISCUSSION For this experiment, we had been assigned to use the method of spooling DNA from a Banana fruit. DNA, or deoxyribonucleic acid, is found in the cells of all living things. It is the master code or blueprint for the organism. During cell division, this code is copied and passed to new cells. DNA also controls all cellular activities through its role in protein synthesis. A filtrate is made of bananas and treated with a buffer containing salt (NaCl) and distilled water. The salt solution acts as a buffer to maintain a constant pH and binds up the positive ions to prevent enzymes (DNA) from chewing up the DNA. The salt shields the negative phosphates of the DNA which allows these ends to come closer so that they can precipitate out of a cold alcohol solution. A detergent is added which causes the cell membrane to break down by emulsifying the lipids and proteins which allows the cell membrane to break apart. The soap was used to dissolve the phospholipid bilayers or the cell membrane around the organelles, and the cells themselves allowing the DNA to be released. The salt breaks up the protein chains that bind the nucleic acids. DNA does not dissolve (is not soluble) in ethanol, and the colder the ethanol is, the less DNA that will be broken down allowing to view the DNA in the test tube. The DNA was separated from the cells by the soap, because it will destroy the cell, and organelle membranes allowing the DNA to be extracted from the cell. The salt breaks the protein chains that bind the nucleic acids and prevent them from being completely separated from the cells. The ethanol doesnt dissolve the DNA so it allow us to view the DNA in test tube that look like a ring of DNA molecules floating in the tube. There is also some error that can cause the result. It is important to not produce bubbles while stirring your extraction mixture. Also when using the filter funnel, do not stir the solution roughly while transferring the solution into the test tube. DNA is only soluble at a pH near physiological levels. The baking soda serves as a buffering system that raises the pH and releases the DNA from bound proteins.

CONCLUSION The experiment objective was to extract and isolate DNA from a banana by following common DNA spooling techniques. The objective had achieved and the result had been observed. Based on the result, we had observed the DNA molecules floating in the tube. The liquid detergent was used to break down the lipid cell membrane. The cells then been broken down to expose the DNA. It is recommended that to not produce bubbles while stirring the extraction mixture because once bubbles form, they are hard to remove, so it's best never to get them in the first place. While transferring the solution into the test tube using filter funnel, do not stir the solution because the filter funnel may break. REFERENCES