Restriction Digestion of DNA and Agarose Gel Electrophoresis

(a) Restriction Digestion of DNA

Objective:
To digest plasmid DNA PBR322 into smaller DNA fragments by using restriction
enzymes.

Apparatus and Materials:

Micropipettes (P-10), Pipette tips, Microcentrifuge tubes, PBR322 plasmid
DNA, Restriction enzyme EcoR1, Restriction enzyme Pst1, Buffer for enzyme,
Ice, Distilled water

Procedures:
1. Microcentrifuge tube was labelled with a permanent marker.
2. Indicated amount of the solution was pipetted into the appropriate
microcentrifuge tube.
3. Pipette tips were changed after each pipetting to prevent contamination
of the DNA, enzymes, and buffer solutions.
Ingredients Single Digestion
Fermentas pBR322 DNA 1 uL
Stock concentration 0.5ug/uL, 100ug

Fermentas Pst1 1 uL
Stock concentration 10u/uL, 5000ug

Fermentas 10x buffer EcoR1 with BSA 1 uL

Top up with double distilled water 7 uL
Total volume of reaction mixture 10 uL

4. Completed digestion mixture was floated in 37°C water bath for 1 hour.
 5. At the end of incubation, the tube was then placed at -20°C.
6. The digest remained frozen until next week’s practical session at which
gel electrophoresis was conducted.

(b) Agarose Gel Electrophoresis

Objective:
To run an agarose gel electrophoresis on digested fragment of pBR322 DNA
and visualize it.

Apparatus and Materials:

DNA ladder, Agarose gel powder, loading dye, gel red stain, Micropipettes (P-
10), Buffer for gel electrophoresis, PBR322 plasmid DNA, Ice, Pipette tips

Procedures:
(A) Preparation of 0.8% agarose gel
1. Calculated amount of agarose powder was added to a conical flask.
2. Appropriate amount of 1x TAE buffer solution was added.
3. Agarose powder was dissolved in buffer by heating the solution in a
microwave oven.
4. The solution was poured into the prepared acrylic tray as described in
section (B) after it has been cooled to between 50°C to 60°C.

(B) Gel Casting and Staining

Gel Casting – Using Casting Dams

1. The casting dams were securely fitted over each end of the tray and
was placed onto a level surface. The dams were fitted so that there
 were no gaps between the sides of the tray and the groove in the
dams to ensure that there was no possibility of gel leakage.
2. Desired comb(s) was placed into the groove(s) in the tray.
3. The cooled agarose solution was carefully poured into the prepared
tray so as not to generate bubbles. Any bubbles that occurred was
smoothed to the edge of the gel and dispersed using a pipette tip.

Gel staining - Using gel red stain

1. 12 µL gel red was pipetted to 200 mL melted agarose gel and was
mixed well.
2. The agarose was allowed to set to ensure that the gel remained
undisturbed.
3. The casting dams and comb was carefully removed and the gel was
transferred on tray to the main tank.

(C) Running the Gel

1. The tank unit was filled with 1x TAE buffer until the gel itself is
completely covered with buffer.
2. 10 µL 1kb DNA Ladder was loaded into the first well
3. Using micropipette with sterile tip, 2 µL loading dye was pipetted
onto parafilm, then 8 µL pBR322 DNA was pipetted to the loading dye
droplet. Solution was mixed well by pipetting up and down and then
loaded into the second well. The uncut pBR322 DNA served as a
control.
4. For single and double digestion mixtures (prepared in previous
week), 2 µL loading dye was pipetted to parafilm, then 8 µL reaction
mixture was pipetted to the loading dye droplet. Solution was mixed
 well by pipetting up and down and then loaded into the subsequent
wells.

Loading Digestion Total Please

Content dye mixture volume ‘tick’
volume volume load into well of
well your
group
Well 1 DNA ladder 10 uL 10 uL
Well 2 Double digestion (Pst 1, EcoR1) 2 uL 8 uL 10 uL
Well 3 Double digestion (Pst 1, EcoR1) 2 uL 8 uL 10 uL
Well 4 Single Digestion (Pst 1) 2 uL 8 uL 10 uL ✓
Well 5 Single Digestion (Pst 1) 2 uL 8 uL 10 uL
Well 6 Single Digestion (EcoR1) 2 uL 8 uL 10 uL
Well 7 Single Digestion (EcoR1) 2 uL 8 uL 10 uL
Well 8 Uncut pBR322 (control) 2 uL 8 uL 10 uL
Well 9 Uncut pBR322 (control) 2 uL 8 uL 10 uL
Well 10 Uncut pBR322 (control) 2 uL 8 uL 10 Ul

5. The lid was carefully placed on the tank and was connected to a
power supply.
6. The gel was allowed to run at about 90 volts until loading dye had
reached near to the end of the gel.
7. The power supply was stopped and switched off.
8. The gel was removed and the bands were visualized under UV light.
9. The gel photo was captured.
Discussion:

Gel electrophoresis is a method used to separate fragments of DNA or

RNA according to their molecular size. The fragments are separated by an
electric field through a gel matrix that contains small pores. [1] The DNA is
digested by restrictive enzymes in specific sites, stained and then pipetted into
the wells. When electric current flows through the gel, DNA moves toward the
positive electrode due to its negatively charged phosphate group. The
migration flow of DNA depends on their molecular weight where the smaller
fragments of DNA moves faster and travels a longer distance than the larger
fragments. Not just that, agarose gel electrophoresis also allows purification of
DNA fragments since the DNA fragments are separated according to their sizes.
The fragment of interest can be obtained this way. [2]

In the first well, DNA ladder is added. DNA ladder is used to help
estimate the size of the DNA fragment. In the second and third well, two bands
can be seen in the gel. This signifies that the DNA molecule is split into two
fragments whereas the smaller fragment is further from the well and the larger
fragment is nearer to the well. According to the DNA ladder, the larger
fragment of the double digestion shows that it is approximately 3000 to 4000
bp in size whereas the smaller fragment is about 750 bp. The pBR322 is 4361
bp in length and restriction sites for EcoR1 and Pst1 is located in the 4359th and
3607th bp respectively. Subtraction of the two numbers is 752 bp which is the
actual length of the smaller DNA fragment and when compared with the
estimated value in the experiment, the values are proximate. For the larger
fragment, subtracting 4361 by 752 is 3609 bp which falls in range of the
approximate value for the larger fragment.
 From the fourth well to the seventh well, only one restriction enzyme is
added. The DNA is said to be in its nicked form, or open circular, as its
molecular weight remains the same but is no longer circular. From the eighth
well to the ninth well, no restriction enzyme is added. Undigested pBR322, or
negative control, are circular plasmids and when they are circular, DNA ladder
cannot be used to estimate the size because the comparison would not be
accurate. Although negative control are of the same size, multiple bands are
observed and the reason for this is due to the different conformation of DNA.
The plasmid DNA exist in 3 forms: supercoiled, open circular and linear. The
predominant form of DNA is supercoiled and when in this form, it is able to
migrate faster and further as it is more compact and thus, undergoes less
friction. [3]

Loading dye is used to increase the density of the sample so that the
DNA sinks into the well. It also contains tracking dyes which migrate in the gel
to help keep track of the process of gel electrophoresis.

Moreover, DNA bands cannot be seen clearly with naked eyes because
they are colourless. Ethidium bromide is the conventional stain used to
visualize DNA bands. But in this experiment, gel red stain is used instead.
Although the gel red stain is more expensive, it is less mutagenic, more
sensitive and more stable compared to ethidium bromide. Like ethidium
bromide, gel red only fluorescent when it binds with DNA under UV light. [4]
However, different types of stains have different wavelengths resulting in
different colours.

TAE buffer is added to maintain the pH of the DNA solution. If a buffer is

not used during the experiment, electrolysis of water molecules would occur
and this would result in the formation of H+ ions. The H+ ions can interact with
the negatively charged DNA, neutralizing it and thus preventing the DNA
fragments from migrating towards the positive electrode efficiently. [5]

Subsequently, factors that influence the migration of DNA are the size of
DNA, the concentration of agarose, the type of buffer and the voltage power.
Smaller fragments travel faster and further in the agarose gel. This is due to
the lower frictional and electrostatic forces compared to the larger fragments.
The DNA molecules moves at a rate of which the distance travelled is inversely
proportional to the logarithm of the number of its base pairs. [6] Next,
increasing the agarose concentration will decrease the pore size of the gel.
Large molecules get entangle in the gel matrix and are unable to travel a far
distance while smaller molecules are able to squeeze through the pores of the
gel. This reduces the migration speed of the larger DNA molecules and enables
the smaller DNA molecules to be separated more efficiently. [7]

A buffer solution is not only used to stabilize the pH of the medium, but
it also provides ions to support electrical conductivity. Different types of buffer
solutions have different ionic strengths. The concentration of buffer also
affects the electrophoresis process. A concentrated buffer has high density of
ions in it. When voltage is applied, the heat generated may be enough to melt
the gel. Naturally, the rate of migration is proportional to the voltage. This
implies that increase in voltage leads to higher migration rate. Similarly with
the concentration of buffer solution, if the voltage is too high, it would cause
the gel to melt, giving rise to smeared and distorted bands. [8]

Next, the enzyme responsible for the initial cutting of DNA molecule is
called the restriction enzyme. Restriction enzymes cut DNA molecules at
specific DNA sequences otherwise known as restriction sites. A restriction
enzyme knows where to cut the DNA sequence because the specific sequence
matches with its recognition site. [9] It also performs multiple cuts, yielding
numerous restriction fragments. The enzyme cuts the DNA molecule in a
staggered way, producing fragments with single-stranded overhangs known as
sticky ends which help assist in the formation of hydrogen bond with
complementary sticky ends. [10]

Gloves are worn throughout the experiment to prevent contamination

of DNA and for protection from hazardous compounds.

Conclusion:

Gel electrophoresis is a method used to study the lengths of DNA molecules.

Restriction enzymes cut DNA in specific places. Smaller and shorter pieces of
DNA move faster and further away from the negative side. Ligase forms
phosphodiester bonds that joins two DNA molecules together.

References:

1. Gel Electrophoresis. Retrieved from

https://www.nature.com/scitable/definition/gel-electrophoresis-286
2. Principles of Nucleic Acid Separation by Agarose Gel Electrophoresis.
Retrieved from http://cdn.intechopen.com/pdfs/35089/InTech-
Principles_of_nucleic_acid_separation_by_agarose_gel_electrophoresis.
pdf
3. Electrophoresis. Retrieved from
http://www.bioinformatics.nl/molbi/SimpleCloningLab/electrophoresis.
htm
4. Ethidium Bromide: The Alternatives. Retrieved from
https://bitesizebio.com/417/ethidium-bromide-the-alternatives-2/
5. The Purpose of the Buffer in Electrophoresis. Retrieved from
https://sciencing.com/purpose-buffer-electrophoresis-6613320.html
6. Agarose gel electrophoresis for the separation of DNA fragments.
Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/22546956
7. Agarose Gel Electrophoresis, Factors affecting Migration. Retrieved from
https://msu.edu/course/css/451/Lecture/PT-
electrophoresis%20(2009).pdf
8. Electrophoresis. Factors Affecting Electrophoresis. Retrieved from
https://www.slideshare.net/magendiramanivinayag/electrophoresis-
and-factors-affecting-electrophoresis
9. Restriction enzyme. Retrieved from
https://www.sciencelearn.org.nz/images/2542-restriction-enzyme
10. Restriction enzymes & DNA ligase. Retrieved from
https://www.khanacademy.org/science/biology/biotech-dna-
technology/dna-cloning-tutorial/a/restriction-enzymes-dna-ligase