University of Balamand

BIOL286 - Molecular Biology Laboratory

Experiments 3-4: DNA Extraction, Quantification, and Separation
Date: 22/2/2024 & 29/2/2024
Submitted by: Jad Farah – Tina Nassar – Cristel Nochahrly
Instructor: Rana Andraos
 I. Introduction

Deoxyribonucleic acid (DNA) stands as one of the most fundamental molecules in

molecular biology, serving as life's genetic blueprint. The elucidation of DNA's structure by
James Watson and Francis Crick in 1953 marked scientific history. The double helical structure
of DNA, with its complementary base pairing of adenine (A) with thymine (T) and cytosine (C)
with guanine (G), provides a remarkable mechanism for the faithful transmission of genetic
information during cellular replication. Since its discovery in the mid-20th century, DNA has
been the focal point of extensive research, revolutionizing our understanding of genetics,
evolution, and the very essence of life itself.
In the field of molecular biology, the extraction and quantification of DNA in laboratory
settings serve as fundamental processes underpinning a vast array of scientific experiments
These techniques are vital in unlocking the genetic information encoded within DNA molecules,
enabling researchers to study the intricate mechanisms governing life at the molecular level. By
extracting and quantifying DNA, scientists gain invaluable insights into genetic variation, gene
expression, disease mechanisms, and evolutionary relationships, among other areas of study.
Whether working with cells cultured in vitro, or tissue samples obtained from organisms,
the extraction process aims to isolate DNA while minimizing contamination and preserving its
integrity. The successful extraction of high-quality DNA lays the foundation for subsequent
analyses.
The quantification of DNA is equally crucial in molecular biology laboratories, providing
researchers with essential information about the concentration and purity of the extracted genetic
material. Accurate quantification ensures the precise manipulation and use of DNA samples in
downstream applications, minimizing experimental variability and maximizing the reliability of
results.
A plethora of techniques are employed for DNA extraction and quantification, each
tailored to specific sample types and experimental requirements. From traditional phenol-
chloroform extraction protocols to modern automated purification systems, researchers have at
their disposal a diverse toolkit for isolating DNA from various sources. Similarly, a range of
quantification methods, including spectrophotometric assays, fluorometric assays, and
quantitative PCR (qPCR), offer flexibility and precision in determining DNA concentration and
purity.

II. Objectives:

1- Extract DNA from the cell line U937 using organic solvents.
2- DNA quantification through spectrophotometry
3- Determine the purity of our DNA sample
4- Separation of DNA by agarose gel electrophoresis

III. Materials

DNA extraction

- Falcon Tubes
- Lysis Buffer or DNA buffer
o EDTA 0.5 M (chelating agent that has a high affinity to Mg2+ in the outer
membrane of the cell, creating pores in it and degrading it).
o Distilled water (solvent)
o Tris HCl, 1M, pH = 8.0 (works as a buffer by providing constant pH at a value of
8)
- 1X PBS (Phosphate buffer saline) to wash the cell surface
- Centrifuge
- Proteinase K (degrades proteins so we can extract pure DNA)
- 10% SDS (strong detergent that denatures and confers the same negative charge to all the
proteins).
- Phenol (organic solvent that will dissolve proteins and lipids)
- Chloroform : isoamyl alcohol (24:1 ratio)
- Hot water bath at 55ºC (optimal temperature for the activity of proteinase)
- Sodium acetate, 3M, pH= 5.2 (increases precipitation of DNA)
- Ice cold isopropanol (precipitates DNA)
- Pasteur pipette
- Eppendorf tubes
- 70% ethanol to remove salts, organic solvents…

DNA quantification

- DNA sample from last week preserved at 4ºC

- Distilled water (solvent used to dissolve DNA)
- Vortex Mixer
- Eppendorf tubes
- Spectrophotometer
- Cuvettes
- Micropipette
- Loading buffer
- Preparation of agarose gel (1%)
o Agarose
o Erlenmeyer flask
o Graduated cylinder of
o Graduated cylinder of 1 L
o Weighing balance
o TAE 50X (Tris-acetate EDTA
- 242g Tris-base dissolved in 800 mL ddH2O
- 57.1 mL glacial acetic acid
- 100 mL of 0.5 M EDTA (pH 8.0)
o Microwave (used to dissolve agarose in the buffer)
o Ethidium bromide (fluorescent intercalating agent between the bases of
nucleic acids).

- Gel separating apparatus

 IV. Procedure:

1) DNA Extraction:
1. Harvest the cells in 15 ml falcon tubes. Centrifuge cultured cells for 10 min at 10°C (1200
rpm).
2. Remove supernatant and re-suspend cell pellet in 1X PBS
3. Wash twice with 10 ml 1X PBS, centrifuging between washes.
4. Resuspend pellet in 10 ml DNA buffer (extraction buffer).
5. Centrifuge cells for 10 min at 10 °C (1200 rpm). Remove supernatant.
6. Add 3 ml DNA-buffer, re-suspend the pellet, directly add 125μl Proteinase K (10 mg/ml) and
400μl 10% SDS and shake gently
7. Incubate overnight at 45°C or 2-3 hours at 55°C.
Steps 1 through 7 were performed by the instructor.
8. Add 3.6 ml of phenol under the hood, spin the falcon tube in the automatic rotator for 3 min.
9. Centrifuge for 10 min at 10°C (3000 rpm).
10. Transfer the supernatant into a new tube (15 ml); measure the volume. Add equal amounts
phenol and chloroform/isoamylalcohol (24:1) in a total amount equal to the volume of the
supernatant.
11. Centrifuge for 10 min at 10°C (3000 rpm).
12. Transfer the supernatant into a new tube (15 ml); measure the volume. Add
chloroform/isoamylalcohol (24:1) in an amount equal to the volume of the supernatant. spin
the falcon tube in the automatic rotator for 3 min; centrifuge for 10 min at 10°C (3000 rpm).
13. Transfer the supernatant into new tube, measure the volume. Add 1/10 volume 3 M sodium
acetate (pH 5.2) and 3 x the volume 100% isopropanol (2-propanol).
14. Shake gently until the DNA is precipitated.
15. Use a Pasteur pipette to transfer the precipitated DNA into a tube containing 1 ml of 70%
ethanol.
16. Centrifuge for 20 min at 14,000 rpm. Air dry.
17. Preserve the DNA at 4 °C

2) DNA preparation:
1. Add 50 μl of distilled water to dissolve the DNA
2. Vortex the sample.
3. Spin the sample.

3) Spectrophotometry:

1. Turn on the UV spectrophotometer and set the wavelengths at 260 and 280 nm according
to the instructions.
2. Exclude the absorbance of the solvent using two blank solutions of distilled water. Insert
the cuvettes into cuvette holders in the sample compartment with the optical (clear) sides
of the cuvettes facing the light path.
3. Take 10 μl from the dissolved DNA sample and add them to a cuvette.
Add 490 μl of distilled water
Vf 500 uL
DF = dilution factor = Vi = = 50
10 uL

4. Insert the sample cuvette and read the absorbance of the sample cuvette against the blank
cuvette at 260 nm then 280 nm. Record the readings.

4) Agarose gel electrophoresis of DNA:

1. Set up the gel-casting unit with appropriate comb.

Preparation of TAE 1X buffer

TAE TAE
50X 1X
y 1000mL

C1 x V1 = C2 x V2
50 x V1 = 1 x 1000
1000 𝑥 1
V1 = = 20 mL of TAE 50X buffer
50
 - Add 20 mL of buffer TAE 50X to a graduated cylinder
- Add to it 980 mL of distilled water

Vf 1000 mL
DF = dilution factor = Vi = = 50
20 uL

(Vf = 1L = 1000 mL of TAE 1X and Vi = 20 mL of TAE 50X)

Preparation of 1% agarose gel

1g agarose 100 mL
xg 150 mL
150 𝑥 1
x= = 1.5 g of agarose
100

- Weigh 1.5g of agarose in an Erlenmeyer flask

- Add to the Erlenmeyer flask 150 mL of 1X TAE
- Dissolve in the microwave for 2 minutes while shaking every 30 seconds
- Cool it down in a water bath at 55ºC

2. Fast-cool the gel-solution-containing flask by use of running cold tap water applied to the
side of the flask and by use of constant swirling.
3. Add 2 µl of 10-mg/ml Ethidium bromide under the hood (because its carcinogenic) per 100
ml of warm agarose gel solution. Allow the gel to solidify for 20 – 30 min at room
temperature.
4. Place the gel tray in the electrophoresis tank with the well-side toward the cathode (−ve
electrode).
5. Cover the gel with 1X electrophoresis buffer to a depth of 1 mm.
6. Carefully remove the comb by slowly and vertically lifting the comb up away from the gel.
7. Carefully load the DNA standard markers along with the DNA samples (mixed with the
loading dye).
8. Cover the lid and connect the current.
9. Stop the electrophoresis. Before the loading dye runs off the gel.
10. Perform autoradiography of the gel to properly visualize the bands.

V. Results

After DNA extraction and purification, some DNA precipitated. Indeed, we obtained a
white clot, a white filament upon adding ice cold isopropanol and sodium acetate. We therefore
conclude that DNA is indeed present in our sample and it has precipitated, which indicates a
positive result for the first part of the experiment.

We then measured the optical density at 260 nm (DNA), and at 280 nm (proteins).

260 nm 280 nm
Optical density 0.1555 0.0985

Table representing optical densities of our sample measured at 260nm

and at 280 nm.

OD 260 nm 0.1555
Ratio = = 1.58
OD 280 nm 0.0985

We then compare this ratio to theoretical values.

Theoretically, if we have a pure DNA:

OD 260 nm
1.7 < Ratio <2
OD 280 nm

The ratio obtained for our sample is 1.58, which is less than 1.7, which indicates that our
DNA is not pure. We will discuss further in the discussion section.

We can also determine the concentration of DNA in our sample.

 [DNA] = OD260 nm x 50 x DF

Vf 500 uL
DF = dilution factor = = = 50
Vi 10 uL

[DNA] = 0.1555 x 50 x 50
[DNA] = 388.75 µg/mL

our sample

Image of autoradiography of the gel electrophoresis

On this gel, we obtained 1 light band of DNA of size 50 kb, which indicates that some DNA is
present in our sample.

VI. Discussion

The spectrophotometer reading of the sample at 260 gives the optical density (OD) of
DNA and is used to calculate concentration of DNA in the sample. The reading at 280 nm gives
us the OD of protein. The ratio OD260/OD280 permits us to quantitatively analyze the purity of
the DNA. If it is less than 1.7 the DNA is contaminated, or even worse there is no DNA only
contaminants. If it is more than 2, the sample is mainly composed of proteins, salts, organic
solvents, and other organic molecules. For the DNA to be considered pure the ratio should be
between 1.7 and 2. The ratio obtained by our spectrophotometer readings is 1.58, which is
smaller than 1.7, meaning that our DNA sample is not pure.
Additionally, the resulting band on the gel can give as a qualitative conclusion about the
purity of the DNA. If the band is prominent, then the DNA is pure. If we quantitatively
concluded that the DNA is not pure, we can further detail the reason. A faint band signifies that
the sample contains small amounts of DNA and contaminants or proteins bound to the DNA,
whereas if there is no visible band on the gel, the sample does not contain any DNA, only
proteins. If there are two observable bands, this means that one of them corresponds to the DNA
and the other to the proteins. According to the autoradiography of the gel, there are no smears in
L5, we can see the formation of a band, so our DNA is not degraded. However, compared to the
rest of the prominent bands, ours is very faint, meaning that there is a small amount of DNA with
contaminants bound to the DNA. These contaminants are possibly histones. To further fortify the
fact that there is some DNA present, we were successful in calculating its concentration with a
value of 388.75 µg/mL
To further explain our results, we can consider the experimental errors that may have
occurred that resulted in an impure DNA sample. After the three consecutive centrifugations,
transfer of the supernatant is a very delicate process. The pipette may have breached the interface
between the supernatant and the pellet during the extraction of the last few milliliters of
supernatant (the aqueous phase that contains DNA, which is a polar molecule). Moreover, after
the formation of the white clot resulting from the addition of ice-cold isopropanol and sodium
acetate, we may have not extracted enough of the visible fibers with the Pasteur pipette and
extracted too much of the surrounding translucent liquid.

VII. Conclusion

The main aim of this experiment was to extract and quantify DNA. First, we extracted
DNA from our sample and removed proteins to obtain a non-contaminated DNA. However, after
measuring the optical densities at 260 and 280 nm, the ratio obtained 1.58 indicates that our
DNA is not pure, which may result from the presence of histones proteins bound to it. The
concentration of DNA in our sample was also determined and shown to be 388.75 µg/mL, which
indicates that we have obtained some DNA in our sample: it contains contaminated DNA which
may result from experimental errors. Finally, after separation with gel electrophoresis, we
obtained a faint band indicating the presence of some DNA in our sample.