8/12/2019 Analy Exp 2

1/4

OBJECTIVE

To extract and isolate DNA from a banana by following common DNA spoolingtechniques

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 oforganisms 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 cloningexperiments. 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.

8/12/2019 Analy Exp 2

2/4

PROCEDURE

100 ml beaker was used to prepare the buffer using 60 ml of tap water, 0.75 g (1/8 tsp) ofsodium 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 hassmashed 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 forminutes.

Filter paper in filter funnel was used and the banana was poured in the buffer mixture intothe 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

8/12/2019 Analy Exp 2

3/4

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 mastercode 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 ismade 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, andthe 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 whilestirring 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.

8/12/2019 Analy Exp 2

4/4

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 theDNA. 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