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Lab Manual-1

This laboratory document provides instructions for purifying DNA from banana cells using household solutions to demonstrate the fundamental steps of DNA purification. It summarizes the history of DNA purification, beginning with Miescher who first isolated DNA in 1869 and developed early purification techniques using salt solutions and proteases. The document outlines a routine protocol for purifying DNA from banana cells using detergent, heat, filtration, and precipitation with alcohol to remove interfering proteins, lipids, and other cellular components without damaging the DNA.

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ozlem
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81 views

Lab Manual-1

This laboratory document provides instructions for purifying DNA from banana cells using household solutions to demonstrate the fundamental steps of DNA purification. It summarizes the history of DNA purification, beginning with Miescher who first isolated DNA in 1869 and developed early purification techniques using salt solutions and proteases. The document outlines a routine protocol for purifying DNA from banana cells using detergent, heat, filtration, and precipitation with alcohol to remove interfering proteins, lipids, and other cellular components without damaging the DNA.

Uploaded by

ozlem
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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BIOLOGY FOR LIFE SCIENCES

LABORATORY

PURIFICATION of DNA
OBJECTIVES
The purpose of this laboratory is to isolate DNA from cells and, in the process, become familiar with
the reagents and steps necessary for DNA purification. You will isolate DNA from bananas using
household solutions to demonstrate the fundamental steps involved.

BACKGROUND
In a cell, DNA acts as the genetic repository of information, and for this reason, scientists often study
DNA to learn more about cellular biology. To manipulate or amplify DNA, scientists often must
remove other cellular components that might interfere with their experiment, such as proteins, RNA
and lipids, without damaging the DNA. The first scientist to purify DNA was a Swiss chemist named
Johann Friedrich Miescher (1844–1895). Miescher was working to isolate intact leukocytes from
used hospital bandages. The term “leukocyte” refers to a broad class of white blood cells, which are
involved in a body’s immune response to infection and disease. His initial attempts were
unsuccessful and yielded a viscous lysate that was impossible to handle.
However, by optimizing the composition of the salt solution in which the bandages were soaked, he
was able to obtain intact cells. He then went on to develop a protocol to isolate intact nuclei from
these cells. From these intact nuclei, Miescher extracted an acid-insoluble, alkali-soluble substance,
which he named nuclein. At the time, scientists believed that cells were made up largely of protein,
but Miescher determined that nuclein was not made up of protein because it was not digested by
protease. Nuclein was later renamed nucleic acid. Today we refer to it as deoxyribose nucleic acid
(DNA) to distinguish it from other types of nucleic acids such as ribonucleic acid (RNA). Miescher
soon demonstrated that nuclein was found in
many other cells. Miescher’s original DNA purification protocol was crude, and thus, the resulting
DNA preparation was not pure, preventing an accurate chemical analysis. One of his main concerns
was protein contamination. Miescher set out to modify his original protocol to obtain pure nuclein
relatively free of interfering protein. He turned to pepsin, a proteolytic enzyme that he extracted
from pig stomachs, to eliminate proteins in the cells’ cytoplasm. With this modified protocol, he
obtained nuclein with sufficient purity and in sufficient quantities to perform chemical analyses and
show that nuclein contained carbon, hydrogen, oxygen and nitrogen but, unlike protein, large
amounts of phosphorus and no sulfur. Since Miescher’s early experiments, much work has been
done to optimize and simplify DNA purification. Today, DNA purification is often considered a
routine laboratory process, and most scientists use existing protocols, which take advantage of the
chemical and physical properties of DNA. These protocols allow scientists to focus their time and
energy on other experimental goals.

MATERIALS
Materials Required
• Detergent solution (10 ml liquid detergent, 3 g of salt, 80 ml of distilled water)
• 60°C water bath
• ice-water bath
• glass funnel
• pipets or graduated cylinders
• test tubes
• ice-cold isopropyl alcohol (or ethanol), kept on ice during the DNA purification
• glass rod or wooden stir stick
• distilled water
• 100 g piece of banana pulp
• Beakers
• 100 ml graduated cylinder
• Filter paper

METHODS
1. Place the falcon tube containing the homogenized banana in a 60 °C water bath for 15
minutes, mixing frequently.
2. Remove the beaker to an ice-water bath for 5 minutes to cool the pulp.
3. Insert a filter paper into a glass funnel and place in a clean beaker.
4. Carefully pour approximately 25 ml of homogenized banana into the filter paper. After 5–10
minutes, you should have at least 5 ml of filtered solution. If the volume is less than 5 ml, add
more homogenized banana into the filter paper.
Precipitating the DNA
5. Tilt the test tube containing 15 ml of ice-cold isopropyl alcohol (or ethanol), and
gently pipetthe banana mixture down the side of the tube so that the mixture forms a
layer on top of the isopropyl alcohol. Do not mix.
6. Incubate the test tube at room temperature for 4–5 minutes or until the DNA
begins toprecipitate. Record the appearance of the DNA in your laboratory
notebook.
7. Insert the glass rod or stir stick into the tube, and slowly rotate it to spool the DNA
onto therod. Carefully remove the glass rod, and observe the purified DNA. Record
the appearance of the DNA in your laboratory notebook.

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