International University – Vietnam National University

SCHOOL OF BIOTECHNOLOGY

GENETICS
Lab 3&4:

DNA EXTRACTION
AND DNA QUANTIFICATION

Instructor: MSc. Tong Thi Hang

Section: Wednesday morning
Group Members:
Trinh Le Hoang Minh – BTBTIU18152
Le Nha Tu – BTBTIU18264
Tran Ngoc Thanh Thu – BTBTIU18226
Nguyen Thi Trang – BTBTIU18248
Nguyen Le Uyen Vy – BTBTIU18284

SEMESTER II (2019-2020)
Ho Chi Minh City, July 15, 2020
 Contribution table

Full name Contribution Signature

percent
Trinh Le Hoang Minh

Le Nha Tu

Tran Ngoc Thanh Thu

Nguyen Thi Trang

Nguyen Le Uyen Vy
TABLE OF CONTENTS

I. INTRODUCTION………………………………………………………………....2
II. OBJECTIVES………………………………………………………………….…..2
III. MATERIALS AND METHOD……………………………………………….…..2
1. Materials………………………………………………………………………..2
a. Extraction of DNA……………………………………………………...….2
b. DNA quantification by gel electrophoresis…………..………………...…3
2. Methods………………………………………………………………………...3
a. Extraction of DNA…………………………………………………………3
b. DNA quantification by gel electrophoresis………….……………………6
IV. RESULTS...………………………………………………………………………...7
V. DISCUSSION………………………………………………………………………9
a. Extraction of DNA……………………………………………………………..9
b. DNA quantification by gel electrophoresis………………………………..…11
VI. REFERENCES…………………………………………………………………...15

1
 I. INTRODUCTION
DNA or deoxyribonucleic acid is the genetic material in humans and almost other organisms.
Most DNA is located in the cell nucleus (nuclear DNA), but a small amount of them can also be
found in the mitochondria (mitochondrial DNA or mtDNA) or in the chloroplast.
The extraction of DNA plays a crucial role in studying genetic causes of disease and for the
development of diagnostics and drugs. It is also essential for carrying out forensic science,
sequencing genomes, detecting bacteria and viruses in the environment, and determining
paternity.
DNA extraction is a routine procedure used to isolate DNA from the nucleus of the cells.
Because of the presence of hard cellulose cell wall of plant cells comparison with animal cells,
the applied method for extracting DNA between them are also different. Breaking the cell wall
and cellular membranes (lysis) is done by using mechanical or nonmechanical methods to allow
access to nuclear material, without its degradation. For this, the initial grinding stage is employed
to break down cell wall material and allow access to DNA while enzymes and chemicals are in
an inactivated state. Once the tissue has been sufficiently ground, it can then be resuspended in
the SDS buffer. To purify DNA, insoluble particles are removed through centrifugation, while
soluble proteins and other material are separated by mixing with chloroform. DNA must then be
precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The
purified DNA is then resuspended and stored in TE buffer or sterile distilled water. The quality
of extracted DNA is checked by staining a sample with ethidium bromide and visualizing under
UV light.
There are many methods to check DNA yield. In this lab, we use agarose gel electrophoresis
which uses electrical field to separate DNA fragments according to their size and charge. The
electrical current creates two opposite charges on the gel: one end of the gel has a positive charge
and the other end has a negative charge. Because of the presence of phosphate groups of DNA,
DNA moves to the positive end of the gel. Smaller molecules migrate through the gel more
quickly and therefore travel further than larger fragments that migrate more slowly and travel a
shorter distance. As a result, the molecules are separated by size and we can estimate the DNA
segment length accurately.

II. OBJECTIVES
After this lab, students should be able to:
● Understand and perform DNA extraction procedure from the plant cells
● Recognize the presence of DNA in the extraction
● Gain knowledge about the gel electrophoresis, the principles of this methods and analyze
quality of extracted DNA

III. MATERIALS AND METHOD

1. Materials
a/ Extraction of DNA

- 500mg plant tissue - Water (sterile)

- Eppendorf - SDS buffer is a composed of:
- Microcentrifuge • Tris-HCl 1M, pH 7.4 (10 ml)

2
 - Absolute Ethanol (ice cold) • EDTA 0.5M, pH 8 (2.5 ml)
- 70% Ethanol (ice cold) • NaCl 5M (2.5 ml)
- Sodium Acetate • SDS 10% (2.5 ml)
- Vortex mixer • 𝑑𝑑𝐻2 𝑂 (32.5 ml)
- Incubator
- Pipette
b/ DNA quantification by gel electrophoresis
- An electrophoresis chamber and power supply.
- Sample combs, around which molten agarose is poured to form sample wells in the gell
- Electrophoresis buffer, usually Tris-acetate-EDTA (TAE), Tris-borate-EDTA (TBE) or
Sodium Boric acid (SB) buffers.
- Loading dye, which contains something condense (e.g. glycerol) to allow the sample to
“fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.
- GelRed, a non-toxic dye used for staining nucleic acids.
- Transilluminator (an ultraviolet light box), which is used to visualize GelRed-stained
DNA in gels.
- DNA 100 bps ladder
- Algarose
2. Methods
a/ Extraction of DNA

Step What to do? Explanation/Notes

1 Put 500 mg plant tissue and 50 µl
cell lysis buffer in Eppendorf then
grind strongly to a fine paste.
2 Add an additional 450 µl of cell
lysis buffer and 20 µl Proteinase K.
Grind a few more time then vortex
tubes for 30 seconds
The cell lysis buffer for this experiment contain
SDS detergent and NaOH, for alkaline lysis. SDS
is strong anionic detergent that can solubilize the
proteins and lipids that form the membranes. It
removes the negative ions from the protein and
destroys its confirmation. Because of loss of
confirmation, the protein loses its structure. The
proteins from the cell membrane get damaged and
the cell gets broken. This will help the cell
membranes and nuclear envelopes to break down
and expose the chromosomes that contain the
DNA.
In addition to removing the membrane barriers,
SDS helps release the DNA from histones and other
DNA binding proteins by denaturing them.
Moreover, the cell lysis buffer also raises the pH to
12 or above, which inhibits DNase (DNase has the
highest activity at low pH)[1].

3
 Grinding is required to break and open the cell to
SDS buffer; otherwise, just a small proportion of
cell will be exposed to SDS and many cell
membranes will therefore not be lysed for further
process [2]. And there is a fact that breaking down a
cell wall helps release the cellular constituents,
which is useful for easy access to the nuclear
material of the plant cells.

Proteinase K is an enzyme that cleaves the peptide

bond in proteins next to the carboxyl group of
hydrophobic amino acid residues (aliphatic and
aromatic) [3].
The proteinase K degrades the protein portion of
these molecules. Further, it degrades histone and
releases DNA from chromatin, deactivates DNAse
protein to protect DNA from the destruction of
DNase.

3 Incubate samples at 60℃ for 15 Incubating the SDS/plant extract mixture at 60oC
minutes ensures a completely good lysis of cells in the
suspension, which can digest proteins, suspend
lipids, break down celluloid material, and make
DNA free. Since 60oC is an optimal temperature
for maximizing the proteinase K, the enzyme can
degrade DNase completely [4].
We incubate for about 15 minutes since this is an
ideal time; if we keep the mixture being incubated
for more than 15 minutes, the DNA might begin to
be broken down.

4 After incubation, centrifuge the

plant extract mixture for 5 minutes
at 12000 RPM (revolutions per
minute)

5 Use a pipette to carefully transfer Chloroform (CHCl3) is a nonpolar solvent

supernatant into the new tubes.
Add 400 µl Chloroform: Isoamyl
Alcohol (24:1) to each tube, gently
mix by inverting the samples 4
times
(hydrophobic) in which nonpolar proteins and
lipids dissolve. Because of the polar characteristic,
DNA is insoluble in chloroform and isolated in the
aqueous phase[5]. Moreover, chloroform provides
good products to get the best quality of DNA and
removes the non-DNA component from nucleic
acid.
A mixture of chloroform:isoamyl alcohol (24:1)
is added to promote the partitioning of lipids,
partially denaturing of proteins, and cellular debris
into the organic phase, separating DNA (leaving
isolated DNA) in the aqueous phase. Whereas, the
pure chloroform itself is less soluble in water and is
less denaturing to proteins, thus only it does not

4
 work well as when it is combined with isoamyl
alcohol.

6 Centrifuge at 12000 RPM for 2

minutes

7 After centrifugation, the sample

split into 3 phases (upper phase,
inter phase and lowest phase).
Using pipette to take the upper
aqueous phase and transfer to the
new tubes

8 Add Sodium Acetate to each H+ of water reacts with PO3– of DNA and makes
Eppendorf. Gently mix by DNA soluble in water. Moreover, DNA is also
inverting the Eppendorf hydrophilic in nature, which makes it easily
dissolve in water. However, Sodium acetate breaks
up into Na+ and (CH3COO)– and react with DNA.
The positively charged sodium ion neutralizes
negatively charged PO3– of the DNA [6]. Therefore,
the function of sodium acetate is to make DNA
become less hydrophilic and keep the concentration
of DNA as much as possible.

9 Add 500µl ice cold absolute DNA is insoluble in alcohols and absolute ethanol
Ethanol to each Eppendorf
10 Invert the tubes slowly several
times to precipitate the DNA
can make DNA less hydrophilic so it won’t be
soluble in water, resulting in the increase of DNA
concentration.
Using ice cold condition also results in high
precipitation: Precipitation is the state when a
molecule goes from a higher energy, dissolved state
to a lower energy, solid precipitate. Therefore, with
ice-cold ethanol where has low temperatures will
make this process faster, more efficient, and with a
higher yield [7].

11 Incubate the tubes at −20℃ for 15 Incubating at -20 degree Celsius involves the
minutes beginning of the Isolation process separating DNA
from the mixture. This increases the amount of the
DNA precipitation as well as removes impurities
from DNA.
12 Centrifuge at 12000 RPM for 2
minutes

13 Carefully discard the supernatant

into the wash cup. Add 500 µl
Ethanol 70% to each Eppendorf to
wash the DNA then resuspend the
tube

5
14 Centrifuge at 12000 RPM for 2
minutes. Remove to supernatant.
Wash the precipitate again with
500 µl Ethanol 70%

15 After the wash, remove Ethanol Air-drying step is for well-removing ethanol,
and allow the DNA pellet to air dry which can interfere with restriction endonuclease
for about 15 minutes digests [8]. It helps prevent the residual ethanol
dripping back onto DNA. Then it is easy for the air-
dried DNA pellet to dissolve in water [9].
16 Add 50 µl of sterile water to
resuspend the DNA

b/ DNA quantification by gel electrophoresis

Step What to do Explanation/Notes
Preparing Agaroses Gel
1 Place the gel into gel mold,
position the comb
2 Prepare 0.8% agarose gel: dissolve In the lab manual, we need 50 ml 1X SB
0.4 grams of agaroses in about 53 buffer. However, in fact, we use 53 ml for this
ml 1X SB buffer solution then heat step. The reason is to make up for the lost volume
with microwave (1 minute/time x 2 because of evaporation. For more specific, when
times) until completely dissolved. we put the solution into the microwave to dissolve
Let the solution cool at 60◦C. the agarose in the 1XSB buffer solution, we may
* It is a good idea to microwaves have lost some water-vapour.
for 30-45 secs, stop and swirl, then We need to let the solution cool down to 60
continue to boil degree Celsius since the solution will solidify
when it is cooled down to 30 or 40 degree Celsius.

3 Add 5 µl of GelRed into 60◦C GelRed is an intercalating nucleic acid stain used
solution. in molecular genetics for agarose gel DNA
electrophoresis. When exposed to ultraviolet light,
it will fluoresce with an orange color that strongly
intensifies after binding to DNA[10].
In the protocol in lab manual, at this step we
would use EtBr; however, in practical, our lab are
guided to use GelRed instead.

4 Pour the agaroses into a gel tray

with the well comb in place.
5 Remove the comb. Place the into Place the gel so that the wells will be closest to the
the gel electrophoresis chamber negative electrode, so that the DNA can move in
and fill gel box with 1X SB buffer the right direction.
Make sure that the buffer will be all covered by
the buffer.
* Loading Samples and Running an Agaroses Gel:
1 Add loading buffer to each of
samples.

6
 2 Load 5 µl of DNA ladder into the
first well of the gel, and then
continue to fill other well with 6 µl
of your samples
When loading your sample into the gel, make sure
that the head of the tip of the pipette is dip into the
buffer but just above the well. Moreover, when
releasing the plunger botton of pipette, make sure
that the tip or your pipette is out of the the buffer.
If not, your sample may be sucked back into the
pipette.

3 Run the gel at 90V, in 35 minutes. Make sure that your sample will not go out form
the gel
4 Turn OFF power, disconnects the
electrodes from the power source,
and then carefully removes the gel
from the gel box.

5 Use UV light to visualize DNA

fragments.

6 Switch on the Transilluminator.

Photograph the gel under UV light.

IV. RESULTS
• DNA extraction.

Our group’s sample Sample from a group at Thursday lab

As shown above, while the sample from Thursday’s group is transparent with white opaque
precipitate inside, our sample have brownish color. The problem may be caused by our
carelessness during the process of washing DNA samples by using “washing solution” - mixture
of chloroform : isoamyl alcohol (24:1). The leaves that are used for extracting DNA may consist
high levels of polyphenol. Since we do not invert it carefully, the “washing solution” do not mix
well with our sample. Therefore, polyphenol does not entirely dissolve in the organic phase and
still stay in the upper phase. The presence of polyphenols gave brown color due to oxidation of
DNA[11] .
7
 • DNA quantification

DNA ladder

Smear

Our group’s samples

As shown above, our group does not observe anything after running gel electrophoresis. This
result indicates that DNAs are not present in loading dye (or the amount of DNA is extremely
small) or we did something wrong in the process of DNA quantification.
*For the extraction of DNA:
We may fail to extract DNA at the grinding step. We made a huge mistake at this step as we
do not turn the leaves up to grind evenly but just pressing the leaves. Therefore, the lower leaves
are not thoroughly crushed, so the cells are not broken and opened. Hence, the SDS buffer cannot
access and lyses the cell membrane, leading to the low amount of DNA. Moreover, as we do not
mix the leaves well while grinding, the cell lysis buffer cannot contact to all the leaves, especially
lower leaves. As a result, the membranes of lower leaves do not break entirely and there is a
little amount of DNA extracted.
Another reason leading to our failure may from step 5. As discussed above, our samples
contain high level of oxidized polyphenol, which are thought to become irreversibly bound to
DNA [12]. Therefore, it makes our DNA sample become “not pure”.
*For quantification by gel electrophoresis:
None of the groups that day succeeded, however, group 4 of my lab moved to Thursday lab
and succeeded in quantifying DNA (but the DNA ladder in Thursday lab was not shown). They
extracted DNA from the same leaves as us, so we think the problem was not with the leaves we
used, but it could have happened when we quantified the DNA.
Since all groups used the same agarose gel, the prepared gel we scaled must have a little
mistake. This mistake could be made by wrong calculation or wrong quantification by us.
Another reason could be from the Transilluminator. Maybe it was not in the good condition, so
it interfered our results.
About the smear at the end of the tube, it can be caused by our poor quality sample of DNA
extraction. Our samples may contain contaminant of RNA. To overcome this problem, we can
8
treat our samples with RNase. Moreover, our DNA samples could be degraded, leading to poor
results, including smeared bands.

V. DISCUSSION
a. Extraction of DNA
1. Why do we use Chloroform as an organic solvent?
Chloroform (CHCl3) is a nonpolar solvent (hydrophobic) in which nonpolar proteins and
lipids dissolve. Because of the polar characteristic, DNA is insoluble in chloroform and isolated
in the aqueous phase. Chloroform makes phase separation of the two liquids since it has a higher
density (1.47 g/cm3) and the lipids and cellular debris within chloroform lie into the organic
phase while leaving isolated DNA protected in the aqueous phase [5].
2. What are the functions of phenol, chloroform and isoamyl alcohol?

Phenol: DNA is insoluble in phenol because phenol is a nonpolar solution. However, protein
has both polar and nonpolar groups present in it because of the long chain of different amino acids.
Different amino acids have different groups present on their side chain. Hence, the folding of the
protein into the secondary, tertiary, and quaternary structure depends on the polarity of the amino
acids. The bonds between amino acids are broken by the addition of phenol and protein denatured.
Ultimately, the protein becomes unfolded by addition of phenol [13].

Chloroform: increases the efficiency of phenol for denaturation of the protein. Here,
chloroform allows proper separation of the organic phase and aqueous phase which keeps DNA
protected into the aqueous phase [13]. Chloroform is also nonpolar solvent and denatures the lipid
and nonpolar proteins.

Isoamyl alcohol: In the phenol-chloroform DNA extraction method, isoamyl alcohol helps
in reducing foaming between interphase. The liquid phase contains DNA and the organic phase
contains lipids, proteins, and other impurities. The precipitated protein denatured and coagulated
between both these phases. This will create the cloudy, whitish- foam between interphase. The
anti-foaming agent, isoamyl alcohol stabilized the interphase by removing the foaming. This
will increase the purity of DNA [13].

3. After inverting gently 4 times, why does it divide into 3 phases? What are the mechanisms
occurring? What are there in each phase?
Inverting the mixture creates the movement of substances in the microcentrifuge, helping them
separate easily. Chloroform itself is the one that is insoluble in the water and have higher
density than water (1.49 g/ml > 1 g/cm3), chloroform with other components that are not
dissolved in the water will be in the organic phase. It separates from the aqueous phase be on
the top [5]. In conclusion, DNA will be in the upper aqueous phase while lipids and nonproteins
will be in the lowest organic phase. Each phase consists of:
Aqueous phase (upper phase): DNA
Interphase: plant debris
Organic phase (lowest phase): proteins, lipids
(Source:
https://www.genetargetsolutions.com.au/product/5prime-
phase-lock-gel/)

9
 4. What is the function of sodium acetate? Why do we use sodium acetate instead of
ammonium acetate?

DNA, a polar molecule, can easily interact with water. H+ of water reacts with PO3– of DNA and
makes DNA soluble in water. Moreover, DNA is also hydrophilic in nature, which makes it
easily dissolve in water. However, a salt (sodium acetate) reacts with DNA in DNA
precipitation. It breaks up into Na+ and (CH3COO)–. The positively charged sodium ion
neutralizes negatively charged PO3– of the DNA [6]. Therefore, the function of sodium acetate is
to make DNA become less hydrophilic and keep the concentration of DNA as much as possible.

Using sodium acetate instead of ammonium acetate because ammonium acetate (with a final
concentration of 2 to 2.5 M) is used if the solution contains nucleotides or oligonucleotides up to
30 base pairs long that should not be co-precipitated. It should not be used if the DNA will be
phosphorylated, because the T4 polynucleotide kinase is inhibited by the ammonium ions [14].
Besides, the presence of ammonium acetate causes more carbohydrate co-precipitation with
DNA, whereas the presence of sodium acetate yields high molecular weight DNA which is a
tight fibrous molecule.

5. Why is absolute ethanol instead of isopropyl ethanol used?

Regarding chemical structure of ethanol and isopropyl ethanol, we have:

(Source: https://i2.wp.com/chemistrycachet.com/wp-content/uploads/2019/03/ethanol-
vs-rubbing-alcohol-on-chemistry-cachet.jpg?resize=820%2C450&ssl=1)

Ethanol: -OH is attached to the first Carbon, Isopropyl ethanol: -OH is attached to the
second Carbon. It is clear that absolute ethanol is more active than the isopropyl ethanol in
terms of reaction order.
6. Why should we use ethanol 70o instead of 60o or 90o ethanol?

Ethanol 70o generally contains alcohols and can be used to wash DNA from proteins,
salts and other contaminants remained. Ethanol 70o means that it contains 70% of alcohol and
30% of water, which can be coagulated when putting it in the fridge while ethanol 90o or
absolute ethanol tends to not coagulate completely. Besides, DNA dissolves in the water
because of its polar characteristic so ethanol 70o has enough concentration to rinse DNA from
other contaminants remained and keep the concentration of DNA as much as possible to the end
(ethanol 60o has 40% of water so the concentration of DNA is less than that of ethanol 70o).

7. After extraction, why do we need to carry out the Isolation process?

10
 The method has to be properly selected to optimize the yield and quality of the DNA
extracted. It also rinses other chemical compounds remaining and makes DNA form be more
visible as a white string.

8. Why do we use H2O instead of TE buffer to store DNA?

TE buffer for DNA storage contains EDTA which is inhibiting DNases and protects the
DNA from degradation. The pH of TE buffer is slightly basic while water has a slightly acidic
pH. Precipitated DNA re-dissolved in high pH solutions like TE buffer. TE buffer has a higher
pH than DNA so it dissolves DNA properly [6]. Alternatively, the DNA can also be dissolved in
water. However, water is the alternative option to store DNA in the short-term.

b. DNA quantification by gel electrophoresis

1. Why does DNA need to be added into low or high pH? Does it charge, and how?

In the neutral pH, almost all of DNA is completely formed in double-helix structure.

Conditions of low or high pH tend to destabilize DNA and may lead to denaturation. Below
pH 5 and above pH 9, the double-helix form of DNA is destabilized because of titration of the
polar groups on the nitrogen bases. The hydrogen bonding between base pairs which is in polar
groups would be broken due to a net charge to polar bonds by ionization.

At high pH, the increase of hydroxide, negatively charged ions, can pull hydrogen ions off
of molecules like the base pairs in DNA. In detail, deprotonation of guanine’s N(1) and
thymine’s N(3) eliminates these as proton donor sites for H-bonding, which leads to their
acquiring a relatively negative charge so that their mutual repulsion increases [15].

At low pH, protonation of adenine’s N(1) and cytosine’s N(3) eliminates these as proton
acceptor sites for H-bonding. Moreover, protonation of all bases leads to their acquiring a
relatively positive charge so that their mutual repulsion increases [15].

2. What is the function of the gel? What is the position that we need to put DNA in a gel
tray?

Gel, a slab of Jello-like material, is involved in gel electrophoresis. Gels for DNA separation
are often made out of a polysaccharide called agarose as the dry and powdered flakes. When the
agarose is heated in a buffer (water with some salts in it) then cooled down, it will form a solid,
slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held
together by hydrogen bonds and form tiny pores [16].

At one end, the gel has pocket-like indentations called wells, in which the DNA samples will
be placed:

(Source: https://cdn.kastatic.org/ka-
perseusimages/ec82418c42e1a4176745273dab3c204bf50cc603.png)

11
 Before the DNA samples are added, the gel must be placed in a gel tray. One end of the box
is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main
body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can
conduct current. The end of the gel with the wells is positioned towards the negative electrode.
The end without wells is positioned towards the positive electrode. The DNA is a negatively
charged molecule because of the phosphate groups in their sugar-phosphate backbone so they
start moving through the matrix of the gel towards the positive pole.
3. Why do we use 0.8% instead of 1.5% agarose gel?

For plasmid agarose gel electrophoresis, a 0.8% - 1.5% agarose gel is prepared as per the
size of the plasmid DNA expected [17].

0.8% agarose gel is good for DNA pieces that range in size from 1 to 25 kb. For smaller
pieces of DNA (200-2000 bp) is 1.5% agarose [18].

The DNA sample of the experiment is large size (genomic) so we have to use 0.8% agarose
gel corresponding to the size of DNA.

4. How to calculate the concentration of agarose in 0.8% agarose gel with 50ml 1X SB
buffer?
To get 0.8% agarose gel, the amount of agarose in 50ml 1XSB buffer is: 0.8% x 50= 0.4g

5. Which are the types of DNA corresponding to the proportion of agarose gel?

(Source:
https://www.thermofisher.com/content/dam/LifeTech/global/brands/Documents/1114/gene
ral-recommendations-dna-electrophoresis.pdf)

Because all DNA fragments have the same amount of charge per mass, small fragments
move through the gel faster than large ones. The smaller DNA is, the larger the agarose gel
percentage is as well as the smaller the size of well is.

6. Why do we replace EtBr by GelRed?

GelRed is an innovative, stable, non-hazardous, and environmentally safe fluorescent nucleic

acid dye. It is an alternative replacing the highly toxic Ethidium Bromide (EtBr) for staining
nucleic acid in agarose gel and polyacrylamide gels. GelRed is very sensitive and can be used
with existing EtBr UV imaging systems [19]. Moreover, it has no change to equipment or optical
settings when replacing EtBr by GelRed.

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 7. Why do we need to remove bubbles on the gel?

It is best to remove all bubbles on the gel because:

• They can cause DNA bands to be distorted: Air bubbles will interrupt with the movement
of DNA during the agarose gel electrophoresis which will lead to inaccurate results as
the positions of different bands will be affected
• Bubbles can cause the loss of conductivity being air of agarose gel therefore make
mistakes such as DNA move poorly in the gel. Once bubbles had solidified, they will
deform wells and affect how the samples in those wells run
• In addition to that, air bubbles formed will also interfere with the electric field provided
in the kit making it to be inconsistent

8. What are the components of the loading dye? The function of each component?

DNA Loading Dye contains: [20]

· Tracking dyes: Bromophenol Blue, Xylene Cyanol FF and Orange G
· Glycerol
· Tris-HCl (pH 8.0)
· EDTA
· Water
Function:

Tracking dyes: for visual tracking of DNA migration during electrophoresis

Glycerol: The presence of glycerol ensures that the DNA in the ladder and sample forms a
layer at the bottom of the well
The EDTA: binds divalent metal ions and inhibits metal-dependent nucleases.

9. What is the difference between loading dye and staining dye?

The difference between loading dye and staining dye is that:

The loading dye is added directly to the DNA samples, the loading dye is used for visual
tracking of DNA migration during electrophoresis. Glycerol is also included in the loading dye
solution which helps prevent the samples from running off the buffer.

The staining dye is mixed with the agarose gel, and the stain binds to the DNA which
increases the bands visibility by making them glow green and allows the DNA to be seen under
the UV light.

10. Could we increase the voltage of the electrophoresis machine? What do we need to notice
in order to use the appropriate voltage?

We can increase the voltage of the electrophoresis machine. The higher the voltage, the
faster the DNA will travel through the gel.

However, we need to notice in order to use the appropriate voltage because turning the
voltage way up means the solution is going to heat up faster => will not be good for the gel as
well as the equipment. Voltages that are too high can possibly melt the gel or cause smearing or
distortion of DNA bands.

The best way to go is use the voltage recommended by the manufacturer of the
electrophoresis machine and stop electrophoresis when the dye reaches the bottom of the gel.
13
 11. If the gel had already been solid and the sample had not been loaded, what would we
have done when Ethidium Bromide (EtBr) had not been added?

Ethidium Bromide (EtBr) is added to help visualize the DNA bands. Without it, it is
impossible to visualize DNA under UV light.

If you forgot to add Ethidium Bromide (EtBr) then we can use EtBr after the electrophoretic
separation. In fact, many people prefer to stain it afterward to avoid contamination of the
electrophoresis equipment. We can reuse the EB for staining for a few weeks either

12. If the gel had already been runned but the loading dye had not been added, what would
we have done?

Adding a loading dye to your DNA before electrophoresis has the effect that the dye binds
to your DNA and increases the density of DNA samples to remain in the bottom of the wells in
the gel. The dye also gives you some hint how far your DNA has run after a certain time. The
dyes migrate in an electric field towards the anode at predictable rates. This enables one to
monitor the electrophoretic process.

Without the loading dye => DNA samples would float out of the wells; we would not know
when to turn the power off and we also would not see bands.

In summary, if the gel had already been run but the loading dye had not been added, then we
can only throw away the samples.

14
VI. REFERENCES

Retrieved 2017, October. Genetics laboratory manual

Brennan, J. (2019, March 02). Components of Lysis Buffers. Retrieved July 05, 2020, from
https://sciencing.com/components-lysis-buffers-8148370.html [1]

Rogers, S., & Bendich, A. (1989, January 01). Extraction of DNA from plant tissues. Retrieved
July 05, 2020, from https://link.springer.com/chapter/10.1007/978-94-009-0951-9_6 [2]

Proteinase K DNA extraction method. (2020, January 10). Retrieved July 05, 2020, from
https://geneticeducation.co.in/proteinase-k-dna-extraction-method/ [3]

Anjum, N. (2015, May 11). In CTAB DNA extraction method after grinding in CTAB, why it is
incubated at 65 oC temperature for almost 30 minutes? Retrieved July 05, 2020, from
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