Experiment no 1

Aim - DNA extraction from the given sample of onion.

INTRODUCTION -
DNA is present in the cells of all living organisms. This procedure is designed to
extract DNA from onion in sufficient quantity to be seen and spooled. It is based
on the use of household equipment and supplies.

Material Required -

 an onion;

 100 mL of a solution of meat tenderizer (0.05 g / mL water) and

dishwashing liquid (0.01 mL / mL water);

 50 mL 95 % ethanol kept in the freezer or on ice;

 ice bath;

 cheesecloth;

 glass stirring rod;

 five (5) test tubes;

 250, 500 and 1000 mL beakers;

 blender;

 scale;

 water bath at 60º C;

 stopwatch.and some chemicals,etc.

Theory - An onion is used because it has a low starch content, which allows
the DNA to be seen clearly. The salt shields the negative phosphate ends of DNA,
which allows the ends to come closer so the DNA can precipitate out of a cold
alcohol solution.
Deoxyribonucleic acid is a molecule composed of two polynucleotide chains that
coil around each other to form a double helix carrying genetic instructions for the
development, functioning, growth and reproduction of all known organisms and
many viruses. DNA and ribonucleic acid are nucleic acids

DNA in Plants. DNA is the hereditary or genetic material, present in all cells, that

carries information for the structure and function of living things. In
the plant kingdom, DNA, or deoxyribonucleic acid, is contained within the
membrane-bound cell structures of the nucleus, mitochondria, and chloroplasts.

PROCEDURE -
 1. Chop the onion into small pieces and place inside the blender container.
 2. Measure out 200 mls (1 cup) of the water. Pour this into a glass and add
one level teaspoon of salt. Microwave the salty water for one minute (High)
then stir well.
 3. Add enough of the salty water to just cover the onion.
 4. Blend the mixture for about 10 seconds.
 5. Pour the onion-salt-water mixture through the strainer and collect the
liquid in a glass. The DNA from the onion is in this liquid.
 6. Measure out 50 mls (1/4 cup) of the onion liquid and pour it into a glass.
 7. Add 2 teaspoons of dishwashing detergent to the glass containing the
50mls (1/4 cup) of onion liquid. Stir very gently.
 8. Measure out 100 mls (1/2 cup) of methylated spirits. Slowly add the
methylated spirits to the glass containing the onion-detergent mixture.
 9. Now wait for the DNA to appear! The DNA is white, rising up from the
bottom of the glass. Be patient as it may take a few minutes to appear.
 Another method for the extracrtion of DNA form the onion

Result- DNA was observerd.

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
  All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided

Experiment no 2
Aim - DNA extraction from the given sample of banana.

INTRODUCTION -
DNA is present in the cells of all living organisms. This procedure is designed to
extract DNA from onion in sufficient quantity to be seen and spooled. It is based
on the use of household equipment and supplies.

Materials Required:
• Extracting DNA from Bananas student handout •
1 large banana
• 3/4 cups distilled water
• 1 teaspoon clear, colorless (i.e., not cloudy) shampoo or liquid soap
containing EDTA
• 1/4 teaspoon table salt
• 15 ml 91% isopropyl (i.e., rubbing alcohol) in 25 ml or 50 ml sealed test
tube; chill the alcohol by placing the test tube in a beaker containing ice
cubes and some water
• Blender or smoothie maker
• 3 16-ounce plastic cups
• tape (optional)
• 2 plastic spoons
• 1 set of measuring spoons and a measuring cup with 1/2-cup markings
 • 1 #4 cone paper coffee filter
• 250 ml beaker
• 1 plastic pipette or medicine dropper
• 1 thin glass rod,etc.

Theory - Cells are the functional units of living things. They reproduce, in
part, by making and passing deoxyribonucleic acid (DNA) from the parent
cell to the offspring cell. All DNA is made up of the same chemical bases,
adenine, thymine, guanine, and cytosine. The order of the bases
determines the proteins the cell makes and the functions the cell performs.
In this activity, students extract DNA (and also some RNA) from bananas.
They see that: • DNA is a component of living and once-living things. • DNA
can be extracted and observed.
Cells reproduce in part by passing deoxyribonucleic acid (DNA) from
parent cells to offspring cells. DNA provides a blueprint for an organism’s
growth and development.
Procedure -
1. Put 1/2 cup of distilled water and one banana into the blender. Blend for
25 seconds, making sure the banana is completely pulverized. Pour the
mixture into a beaker.
2. Mix 1 teaspoon of soap with 1/4 teaspoon of salt in a plastic cup. Add 2
tablespoons of distilled water. Stir gently to avoid creating a foam. Continue
for a few minutes until the soap and salt are dissolved
. 3. Add 2 tablespoons of the banana mixture to the cup containing the soap
solution. Use a spoon to stir the mixture for at least 10 minutes.
4. Insert a filter into a clean plastic cup so it does not touch the bottom of
the cup. If necessary, tape the sides of the filter to the cup.
5. Pour the mixture from step 3 into the filter. After 10 minutes, some
liquid, called the filtrate, should have collected in the bottom of the cup.
Gently stir the mixture in the filter and let it sit for another minute. Remove
the filter and set it aside.
6. Get a test tube of cold alcohol. Use a pipette or eyedropper to collect your
filtrate. Add it to the alcohol.
 7. Place the test tube with the alcohol and filtrate in a beaker or test tube
holder. Let it sit undisturbed for about four minutes. Do not shake. The
white material coming out of solution as a precipitate is DNA.
8. Dip the glass rod into the tube, slowly rotating it to spool out the
banana’s DNA.

Result – DNA was observerd.

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided.

Experiment no 3
Aim - DNA extraction from the given sample of Tomato

INTRODUCTION -DNA is present in the cells of all living organisms. This

procedure is designed to extract DNA from onion in sufficient quantity to be seen
and spooled. It is based on the use of household equipment and supplies.
Material Required
- Tomatoes
- Table sal
- Lemon juic
- filtrated
- Washing-up liquid (colorless)
- Kitchen knife
- Mortar
- Coffee filter
- Alcohol (ethanol, 96%, -20°C)
- Test tube with cork plug – Toothpick

Theory -he genomes of the two plants have 92 percent of their DNA in

common, the tomato researchers report. The main difference is that the
potato is thought to have a handful of genes that direct the plant's energy
away from producing fruit and into the generation of tubers.

Procedure -
1. Cut half of a tomato into small pieces with the kitchen knife. Put them
into the mortar.
2. Prepare an extraction buffer: 0.5 g table salt 25 ml filtrated lemon juice 5
ml washing-up liquid (colorless) 20 ml water
3. Pour the extraction buffer into the mortar and mash the tomato pieces
thoroughly for about a minute .
4. Take the content of the mortar and let it drop through the coffee filter into
a clean glass.
5. Take 1.5 ml of the filtrated liquid and pour it into a test tube.
6. Add 1.5 ml water and cover gently with a layer of 6 ml freezing cold
alcohol)
7. Hold the test tube quiet for a short time. Alcohol is less dense than water,
so it floats on top of the water layer.
8. The DNA is now visible as a „white ball“ between ethanol and water.
Look for clumps of white stringy stuff where the water and alcohol layers
 meet. If you want, you can try to grab the DNA ball with the toothpick and
take it out of the test tube.

Result –DNA was observerd.

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided.

Experiment no 4
Aim - DNA extraction from the given sample of orange.

INTRODUCTION -
DNA is present in the cells of all living organisms. This procedure is designed to
extract DNA from onion in sufficient quantity to be seen and spooled. It is based
on the use of household equipment and supplies.
Materials required -

 Fruit – Orange
 5 g washing up liquid
 2 g salt
 100 ml tap water
 100 ml of ice cold alcohol (isopropyl alcohol can usually be found at the
pharmacists); put in a freezer for at least 30 mins before starting the
experiment
 Access to hot water - about 60 °C
 Sieve or coffee filter paper
 Two glass beakers (or old jam-jars)
 Several bowls of different sizes, including a large bowl for making a water
bath
 A paperclip
 Safety spectacles - if desired

Theory - Cells are the functional units of living things. They reproduce, in
part, by making and passing deoxyribonucleic acid (DNA) from the parent
cell to the offspring cell. All DNA is made up of the same chemical bases,
adenine, thymine, guanine, and cytosine. The order of the bases
determines the proteins the cell makes and the functions the cell performs.

Proceduce –
Step 1: Mash up the fruit of your choice in a bowl. Bananas, kiwis and strawberries
all work well. (Remove the skin of the bananas and kiwi, we just want the insides!)

Step 2: In a separate bowl, mix the washing up liquid, salt and tap water. Stir
gently trying to avoid making too many bubbles in the mixture. This is your
extraction buffer.
Step 3: Add the fruit to the extraction buffer and mix again. Mash your fruit
sample as much as you can, but again, try to avoid making too many bubbles.

Step 4: Make a water bath with a temperature of about 60 °C. (A large washing up
bowl works well for this.) Leave the fruit extraction mixture to incubate for 15
minutes.

Step 5: After 15 minutes, filter your fruit mixture through a fine sieve or coffee
filter. This will remove all the solid material that you don’t want. You should be
left with a clear(ish) liquid.

Step 6: Take the ice cold alcohol and very slowly, drop by drop, pour it down the
inside of the container with your fruit mixture. What you want to do is produce a
layer of the alcohol floating on top of the fruit mixture.

Step 7: At the interface between the alcohol and the fruit mixture, you should see a
white cloud-like substance forming. Use a hook (a bent paperclip would work) to
slowly draw the DNA up and out of the solution.

Result – DNA was observerd.

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided.
 Experiment no 5
Aim –To determine the amount of DNA in the given
sample of DPA method.
Principle: The deoxyribose in DNA in the presence of acid forms β-
hydroxylevulinaldehyde which reacts with diphenylamine to give a blue
colour with a sharp absorption maximum at 595nm. In DNA, only the
deoxyribose of the purine nucleotides react, so that the value obtained
represents half of the total deoxyribose present.
) Materials required:

Equipments:
• Spectrophotometer

• Water bath

. Chemicals/reagents:

• Standard DNA solution (0.25mg/ml)

• Diphenylamine reagent

• DNA sample in saline citrate buffer

• Saline citrate buffer (0.15M NaCl, 0.015M sodium citrate, pH 7.0)

• Glacial acetic Acid

• Concentrated H2SO4

• Ethanal

. Glasswares and others:

• Test tubes

• Pipettes

• Graduated cylinder
b) Procedure:

Preparation of reagent: Dissolve 1.5g diphenylamine in 100ml of glacial

acetic acid. Add 1.5ml of conc H2SO4.

Store the solution in a dark glass bottle.

On the day of use, prepare a fresh solution of ethanal (1ml) in dH2O (50ml).

Add 0.5ml of this solution to each 100ml of the diphenylamine solution.

Caution: Wear eye protection and use a fume cupboard when preparing
this reagent. Diphenylamine is harmful if ingested or inhaled and may
irritate skin or eyes if it comes into contact with them.

c) Assay:

1. Prepare a series of dilutions of standard DNA (0.25mg/ml) in saline

citrate buffer to give a concentration of 50-500µg/ml.

2. Prepare all the samples in triplicate.

3. To 2ml of each dilution of blank, standard and unknown add 4ml of

diphenylamine reagent and mix. Tube1 is used as blank and tubes 2
through 7 are used for construction of a standard calibration curve for
DNA. Tubes 8-11 are for unknown samples. (Table1)

4. Incubate all the tubes in boiling water for 10 min.

5. Cool the tubes and read the absorbance at 595nm against the blank.

6. Construct a standard curve of absorbance A595 vs. quantity of DNA and

then calculate the concentration of unknown DNA dissolved in the saline
citrate solution.

Observation table –
 Calculation: Determine the amount of DNA in the unknown sample by
plotting a standard curve of A595 on Y-axis and µg of DNA on X-axis

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided

Experiment no 6
Aim –To isolate genomic DNA from animal tissue

INTRODUCTION -
A simple technique for the isolation of very high molecular
weight genomic DNA from animal tissues and cells is described.
The method involves rapid isolation of nuclei and their embedding in
agarose beads followed by extraction of lipids and proteins with SDS.

Basic considerations
DNA extraction is required for a variety of molecular biology applications.
Figure 1 lists the basic steps involved in all DNA extraction methods. Many
commercial kits are available to isolate DNA from a variety of biological
materials The sensitivity of polymerase chain reaction (PCR) detection has
been shown to be different for various DNA kits Therefore, selecting the
best methodology for your application is crucial.

Choosing the correct DNA extraction kit can save crucial time on optimization and
execution of the experiment. Factors to be considered for selecting a kit include:
1. Sample origin: Different kits are used to extract material from specific
sources, including human tissues, blood, hair, rodent tissues, leaf tissue, bacteria,
yeast, fungi, insect, stool, body fluids, spores, soil, clinical samples (e.g., biopsy
 samples, fine needle aspirates), forensic samples (e.g., dried blood spots, buccal
swabs), and fingerprints
2. Preparation method: Sample preparations can be: fresh or previously frozen
cell pellets, paraffin-embedded or formalin-fixed tissue sections, frozen tissue
sections, ethanol-fixed cells, Oragene®-preserved samples, and samples from
forensic sources which might contain very limited material
3. Intended use: The quality and purity of the DNA provided by the kit should
be suitable for the intended downstream application, which could be sequencing,
fingerprinting, PCR, quantitative PCR (qPCR), Southern blotting, random
amplification of polymorphic DNA (RAPD), amplified fragment length
polymorphism (AFLP), and restriction fragment length polymorphism (RFLP)
applications, restriction endonuclease digestion, or the preparation of shotgun
libraries.
4. Humic content: If the sample has humic content such as compost, sediment
and manure, a kit/method that removes humic substances should be used, as they
can inhibit downstream applications like PCR.
5. Sample quantity: The kit to be used depends on the size of the sample being
analysed. For example, the number of cultured mammalian cells (105-107) and
bacterial cells (106-1011), the weight of human tissue, plant tissue or soil,, the
volume of blood, or even trace DNA samples from a crime scene.
6. Yield: the desired or expected amount of DNA to be purified from the
sample. This is dependent upon the sample as well as the downstream
applications
7. Simplicity: The kit operation depends on the experience of the user, and the
degree of control desired over each stage of the sample processing.
Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided

Experiment no 7
Aim – to extract and analyze genomic DNA from
bacterial cells following solution based methods.
MATERIALS INCLUDED This kit has enough materials and reagents for 24
students (six groups of four students.)

• 6 vials E.C. Cell Pellet

• 4 vials Sterile Water • 1 vial Protease: Dry Proteases

• 1 bottle DNA Release Buffer

• 1 bottle Precipitation Solution

• 1 bottle DNA Salt Solution

• 60 2ml Centrifuge Tubes SPECIAL HANDLING INSTRUCTIONS

• All reagents can be stored at room temperature The majority of reagents and
components supplied in the BioScience Excellence™ kits are non toxic and are
safe to handle, however good laboratory procedures should be used at all
times.  This includes wearing lab coats, gloves and safety goggles.

ADDITIONAL EQUIPMENT • Waterbath or beaker and

thermometer
• Mini Centrifuge
• Agarose Gel Equipment (optional)
• DNA Loading Buffer (optional)
• 70% Ethanol (Optional

OBJECTIVES
• Isolate bacterial genomic DNA.
BACKGROUND - DNA, deoxyribonucleic acid, is the molecule of
life.  Every living organism has DNA in each cell of the organism and
each molecule of DNA carries the blueprint for that organism.  The
DNA molecule is also responsible for heredity, passing on genetic
information from parents to child.
DNA molecules are large strands or chains of small molecules known
as nucleic acids, which are localized in the nucleus of a cell.  This kit
allows students to break open bacterial cells and their nuclei to
release the genomic DNA using a protease to digest away the cell and
nuclear walls.  Once released, the genomic DNA is visualized by the
addition of a precipitating solution (alcohol) and high salt, which
causes the DNA to precipitate and become visible.  
  TEACHER’S PRE EXPERIMENT SET UP
1. Heat a waterbath or heating block to 50‐55°C is required for
efficient release of the genomic DNA. A beaker with warm water and
a thermometer can also be used.
2. Add 1ml Sterile Water to each vial of bacterial E.C. Cell
Pellet.  Vortex or vigorously shake until a homogenous mixture, with
no lumps, forms. Supply each group with one vial.
3. If extra vials are available, aliquot the reagents for each group as
indicated in the following section.
4. Prior to the commencement of the experiment, add 0.5ml Sterile
Water to the vial of dry protease to rehydrate.  Mix by inverting the
vial several times until a white suspension is visible.  This solution can
be stored frozen for up to 1 week
.
   MATERIALS FOR EACH GROUP
• 1 vial E.C. Cell Suspension
• 0.8ml DNA Release Buffer
• 80μl Protease  
• 0.5ml DNA Salt Solution
• 4ml Precipitation Solution  
• 8 2ml Centrifuge Tubes    
PROCEDURE-
1. Label two 2ml Centrifuge Tubes with your name and transfer
0.2ml E.C Cell Suspension, a suspension of bacteria, to one of your
tubes.
2. Add 0.2ml DNA Release Buffer to the tube containing the Bacterial
Suspension.   Invert the tube several times to slowly mix.  The DNA
Release Buffer breaks open the bacterial cells releasing the DNA.
 3. Add 0.02ml Protease to the tube to digest and remove the cellular
material and protein and release the genomic DNA.
4. Close the cap.  Briefly mix by inverting the tube 5‐6 times and then
place in a 50‐ 55°C waterbath or heating block for 1 hour.
5. After 1 hour, add 0.1ml DNA Salt Solution to the tube and mix by
inverting the tube several times.  The salt solution aids in the
precipitation of the DNA
. 6. Centrifuge the tube for 5 minutes at 5,000xg to pellet the cell
debris.  Transfer the supernatant to your other labeled tube. 
  7. Add 0.8ml Precipitation Solution, close the tube and, whilst
watching, slowly invert the tube several times to mix. White DNA
strands may appear.
OPTIONAL:
1.The genomic DNA can be visualized on an agarose gel.  Follow the
steps below to prepare genomic DNA for agarose electrophoresis. 1.
OPTIONAL: To pellet the DNA centrifuge the tube at 14,000rpm for
10 minutes. A tight white pellet should be visualized.
2. OPTIONAL: Remove the Precipitation Solution and wash the pellet
with 0.5ml 70% ethanol and centrifuge as before.  Remove the 70%
ethanol and leave the open tube at room temperature for 10‐15
minutes to dry.  Resuspend in 30μl water and load 10‐20μl on a 1%
agarose gel to visualize the genomic DNA.

Result- The DNA Release Buffer is responsible for breaking open

the bacterial cells to release the genomic DNA into the solution.
Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided
 Experiment no 8
Aim – Extraction of high-quality genomic DNA from different plant
orders applying a modified CTAB-based method

INTRODUCTION -
Reliable measurement of DNA concentration and purity is
important for almost all molecular genetics studies. Different
plant species have varying levels of polysaccharides, polyphenols,
and other secondary metabolites which combine with nucleic
acids during DNA isolation and further affect the quality of the
extracted DNA. The current extraction protocol is based upon the
conventional cetyl trimethylammonium bromide (CTAB) method
with further modifications for the extraction of DNA from variable
plant seeds and crops belonging to seven different orders. The
principle modifications currently employed for DNA extraction
involved the use of higher CTAB concentration and higher levels
of 2-β-mercaptoethanol. Additionally, higher concentrations of
sodium chloride and potassium acetate were added
simultaneously with absolute ice cold isopropanol for the
precipitation of DNA free from polysaccharides.

Material and reagents Requried -

 3× extraction buffer containing: 3% CTAB (w/v), 1.4 M NaCl, 0.8 M

Tris-HCl pH 8.0, 0.5 M EDTA pH 8.0 (autoclaved)
 0.3% 2-β-Mercaptoethanol.
 Chloroform:isoamyl alcohol (24:1 v/v).
 6 M NaCl
 3 M potassium acetate
 Ice cold 100% isopropyl alcohol
  70% ethanol
 1× TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0,
autoclaved).
 Agarose (molecular grade)

Modified DNA extraction protocol

 Preheat the 3× extraction buffer in water bath at 65 °C. Add 0.3%

2-β-mercaptoethanol to the 3× CTAB extraction buffer
immediately before use.
 Grind 50 mg of plant samples into powder in liquid nitrogen using
pre chilled mortar and pestle. While still in the mortar, add 800 μl
of the preheated 3× CTAB extraction buffer to the grinded plant
samples and swirl gently to mix using the pestle.
 Transfer the sample mixture to a 2-ml microcentrifuge tube,
incubate in water bath at 60–65 °C for 1 h, mix gently every 20 
min by inverting the tube for 20 times each, then cool down to the
room temperature.
 Add an equal volume of chloroform:isoamyl alcohol (24:1 v/v) and
mix by slight inversion.
 Centrifuge at 13,000 rpm for 15 min at room temperature (RT).
 Using a wide bore pipet, carefully transfer the upper aqueous
phase, which contains the DNA, to a new 1.5-ml eppendorf tube.
 Repeat the extraction steps when necessary until the upper
aqueous phase is clear.
 Estimate the volume of the aqueous phase (approximately 700 μl)
then add half this volume (350 μl) of 6 M NaCl and mix well.
Successively, add 1/10 the volume (70 μl) 3 M potassium acetate
and simultaneously mix with 500 μl ice cold 100% isopropyl
alcohol (approximately two thirds the volume of the aqueous
phase). Invert gently to precipitate DNA until the formation of
DNA threads.
 Incubate at − 20 °C for 30 min.
 Centrifuge at 13,000 rpm for 5 min, discard supernatant.

 Invert the tube containing the DNA pellet on tissue paper to

complete draining off the supernatant.
 Wash DNA pellet with 500 μl of 70% ethanol and invert once (to
dissolve residual salts and to increase purity of the DNA).
 Centrifuge at 13,000 rpm for 5 min.
  Discard 70% alcohol from tubes. invert the on filter paper, and
allow tubes containing pellet to air dry at room temperature for 15 
min.
 Re-suspend the DNA pellet in 50 μl 1× TE buffer. Incubate the
DNA at 50 °C for 1 to 2 h to ensure complete re-suspension.
 Store at − 20 °C till further use.

Quantitative and qualitative analysis of DNA extracted by established CTAB method and
modified protocol

DNA concentration, purity, and quality

DNA concentration was determined spectrophotometrically at 260 nm (A260)

absorption using NanoDrop1000 (Thermo Scientific). Purity of DNA from
protein and polysaccharide contamination was assessed by estimating the
absorbance ratio at A260/A280 and A260/A230 respectively. The quality of the
extracted DNA using both protocols was also evaluated by electrophoresis
separation for all DNA samples on 0.8% agarose gel stained with ethidium
bromide (1 μg/ml).

DNA digestion analysis

HindIII restriction enzyme was used to digest the DNA samples according to
the procedure of Fang and colleagues). Approximately 20 μg of genomic DNA
was digested separately for 1 h at 37 °C with HindIII restriction enzyme
(Amersham Pharmacia Biotech. UK Ltd). All stained electrophoresis
separation matrices for PCR amplification and both extracted and digested
DNA samples were resolved by SYNGENE Bio Imaging Gel Documentation
System

Results
Applying the present standardized method, the extracted DNA concentrations
varied with the different plant species used in the present work . The yield of
isolated DNA ranged from 2.238 ηg/mg of seeds in case of Cucurbitales
maxima to 24.957 ηg/mg of seeds in the case of Lupinus lupinus. The other
classical CTAB method employed also produced comparable range of DNA
concentration yet with less purity in most cases. Most of DNA samples
extracted by the original CTAB method had A260/A280 ratio below 1.8, while the
A260/A280 ratios ranged from 2.08 to 2.23 in DNA samples extracted by our
modified protocol
 Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided

Experiment no 9
Aim – To quantitatively analyze the DNA sample by agarose gel
electrophoresis.
 Introduction
Nuclear morphology changes characteristic of apoptosis appear within the
cell together with a distinctive biochemical event: the endonuclease-
mediated cleavage of nuclear DNA. In fact, formation of DNA fragments of
oligonucleosomal size (180-200 bp) is an hallmark of apoptosis in many cell
types.

The present protocol provides a method for DNA separation of fragmented

and intact DNA fractions and for their analysis by agarose gel
electrophoresis. In apoptotic cells specific DNA cleavage becomes evident
in electrophoresis analysis as a typical ladder pattern due to multiple DNA
fragments. However, although this protocol is simple and generally able to
provide good results, it is only qualitative because of its limitations in DNA
recovery and solubilization. In order to obtain a cleaner DNA, other methods
for DNA preparation are required (in some cases use of proteinase K for
deproteinization is recommended).

Theory -

Agarose Gel Electrophoresis :

Description- An electrophoresis technique that is used to separate DNA
fragments by size. Negatively charged DNA fragments are separated in an agarose
gel bed by subjecting them to an electric field. The gel is stained so that the DNA
bands can be visualized. The DNA fragment sizes are determined by comparison
to a set of size standards

Uses:
Quantitative and Qualitative Analysis, Characterization, Purification

• Determination of sizes of DNA fragments when compared to standards

• Evaluation of purity of DNA after DNA isolation (DNA and RNA contaminants
can be observed)

. • Determination of DNA size following restriction enzyme digestion.

• Purification of DNA fragments after separation by recovery from the gel.

• Separation of DNA fragments prior to further characterization and analysis (for

example Southern Blotting).

Detailed Description:
Agarose gel electrophoresis is a wonderful tool, and the workhorse of the
Biotechnology lab! The theory is simple. DNA molecules are long polymers, and
the size of the strand is proportional to its negative charges because of the
phosphate backbone. The longer the DNA fragment, the greater its charge. Thus,
when placed in a semi-permeable buffered media, DNA will migrate by size, in a
rate roughly proportional its charge to mass ratio, toward the positive electrode.
Fragments will separate during the course of the gel run, with larger fragments
migrating more slowly. Following the separation, which is usually monitored by a
tracking dye such as bromophenol blue, the gel is stained, the bands visualized,
and a photo or digital record of the gel made. Analysis of a gel will always elicit a
cry or excitement, or a sigh of disappointment. Gels are easy to run, but it is also
easy to make mistakes. Many scientists have pieced together broken gels after
they dropped the slippery gel on the floor.
 The sizes of the unknown DNA bands are determined by comparing them to a set
of DNA markers, frequently referred to as “ladders” because of the rung-like
appearance in the gel lane. A number of markers, in various sizes, are available
commercially.

The separation is dependent on the type of agarose gel, the concentration of

agarose used to make the gel, the buffers, amount of voltage used, temperature
and the size of the DNA. A typical gel would be 1% agarose in a buffer such as Tris-
Acetate/EDTA, run at 80 volts for 60 minutes. Higher percentage gels are used to
separate smaller fragments. Specialty gels are used to separate very small DNA
pieces, or for other applications, such as using low-melt gels for recovery of DNA.
Lower voltages, coupled with longer running times,

provide optimum resolution, such as that required for Southern Blots or forensic
applications. Pulsed-field electrophoresis can be used to separate very large DNA
fragments. The most common stain is ethidium bromide, which intercalates into
the doublestranded DNA (and some RNA) strands. With ultraviolet light and
ethidium bromide stain, nanogram levels of DNA can be observed. Ethidium
bromide is a mutagen,

 so other safer stains have been developed, which are often used in
teaching labs. Most DNA is double stranded, but single stranded DNA and
RNA can also be separated by gel electrophoresis. Intrastrand base-pairing
will cause anomalous migration of the fragments, useful for studying
polymorphisms.

Materials
 Cell suspension at 1-5x106 cells/ml in complete RPMI medium (A1)
 TTE solution: TE buffer pH 7.4 (A1) with 0.2% Triton X-100 (store at 4°C)
 NaCl 5M, ice cold
 Isopropanol, ice cold
 Ethanol at 70%, ice cold
 TE buffer pH 7.4 (A1)
 Loading buffer 10x (A1)
 TBE buffer for electrophoresis (A1)
 Ethidium bromide solution (A1)
 Electrophoresis-grade agarose
 DNA molecular weight markers
 Refrigerated cell centrifuge (A3)
 Microfuge (A3)
 Heating block (A3)
 Gel electrophoresis apparatus (A3)
 DC power supply (A3
 Polaroid Camera + films (A3)
Methodology
 
1. Dispense 0.5 ml of cell suspension (no less than 5x105, otherwise DNA
will not be detectable by photography of ethidium bromide stained gel, and
no more than 5x106, to avoid difficult handling of too high amounts of
insoluble DNA) in tubes labeled B (bottom).
2. Centrifuge cells at 200xg at 4°C for 10 min.
3. Transfer supernatants carefully in new tubes labeled S (supernatant).
4. Add to the pellet in tubes B 0.5 ml of TTE solution and vortex
vigorously. This procedure allows the release of fragmented chromatin from
nuclei, after cell lysis (due to the presence of Triton X-100 in the TTE
solution) and disruption of the nuclear structure (following Mg++ chelation
by EDTA in the TTE solution).
5. To separate fragmented DNA from intact chromatin, centrifuge tubes B at
20,000xg for 10 min at 4°C.
6. Carefully transfer supernatants in new tubes labeled T (top).
7. Add to the small pellet in tubes B 0.5 ml of TTE solution.
8. Add to the 0.5 ml volume present in tubes B, S and T, 0.1 ml of ice-cold
5M NaCl and vortex vigorously. The addition of the salt should be able to
remove histons from DNA.
9. Add to each tube 0.7 ml of ice-cold isopropanol and vortex vigorously.
10. Allow precipitation to proceed overnight at -20°C. This step can be
shortened by putting samples in a bath of ethanol/dry ice for 1 hr.
11. After precipitation, recover DNA by pelleting for 10 min at 20,000xg at
4°C.
12. Discard supernatants by aspiration or by rapidly inverting tubes and
carefully remove any drops or fluid remaining adherent to the wall of the
tube with a paper towel corner. This can be a critical step because the pellet
could be loosen and transparent, hard to be seen.
13. Rinse the pellets by adding to each tube 0.5-0.7 ml of ice-cold 70%
ethanol.
14. Centrifuge tubes at 20,000xg for 10 min at 4°C.
 15. Discard supernatants by aspiration or by rapidly inverting tubes.
Carefully remove any drops or fluid remaining adherent to the wall of the
tube by inverting tubes over an absorbent paper towel for 30 min. Let air dry
the tubes in upright position for at least 3 hr before proceeding.
16. Dissolve DNA by adding to each tube 20-50  l of TE solution and
place the tubes at 37°C for 1-3 days. The redissolution of DNA may be a
crical step, in fact it depends on the DNA quantity and size present in the
samples. Thus, the non-fragmented DNA contained in the B tubes, may
need higher volumes of TE and longer incubation times in order to be
resuspended.
17. Mix the samples of DNA with loading buffer by adding 10x loading
buffer to a final concentration of 1x. The addition of loading buffer to
samples allows to load gel wells more easily and to monitor the run of
samples.
18. Place samples in a heating block at 65°C for 10 min and immediately
load 10-20  l of them to each well of a standard 1% agarose gel containing
ethidium bromide 0.5 mg/ml. Appropriate DNA molecular weight markers
should be included. Ethidium bromide is a potential carcinogen: wear gloves
and handle with care.
19. Run the electrophoresis in standard TBE buffer after setting the voltage
to the desired level. During electrophoresis it is possible to monitor the
migration of samples by following the migration of bromophenol blue dye
contained in the loading buffer.
 20. Stop the electrophoresis when the dye reaches about 3 cm from the end
of the gel.
 21. To visualize DNA, place the gel on a UV transilluminator and take
photos of the gel. Wear eye and skin protection when UV are on.

Result -
The assay of DNA agarose gel electrophoresis provides good results for the
definition of cell apoptosis. A typical ladder pattern of DNA fragmentation
should be observed in most apoptotic cells.

Precaution –
 Correct handling & storage of starting material.
 All the equipments should be steril.
 All the steps should be performed gently.
 Measuring should be accurate
 Human error should be avoided