Lab Journal

Lab 2- Visualization and Analysis of Plasmid DNA using

Agarose Gel Electrophoresis.
Genetic Engineering
Group 1

Materials- 20220901008
Reagents and their role- 20220901004
Principle- 20220901001
Protocol- 20220901005
Observation and Result- 20220901003
Discussion- 20220901009
Biorender- 20220901010
Objective: Visualization and analysis of plasmid DNA using gel electrophoresis.

Materials and Methods-

Glasswares:
1] Conical flask
2] Beakers

Plasticwares:
1] Pipette and tips
2] Gel tray
3] Buffer tank
4] Comb
5] Lid
6] Gloves

Equipments:
1] Hot Plate
A hot plate is a lab device that is used are for heating samples or solutions. It is
commonly used in various scientific and chemical laboratories.
2] UV transilluminator
A UV transilluminator is an instrument that emits ultraviolet (UV) light to
visualize nucleic acids, such as DNA or RNA, that have been separated by gel
electrophoresis.
3] Anode (red) and Cathode (black)
The anode (red) and cathode (black) are terms used to describe the two
electrodes in an electrophoresis setup, which is a common laboratory
technique for separating charged molecules like DNA, RNA, and proteins based
on their size and charge.

Chemical reagents and their role-

1. Agarose Gel (1 g Agarose gel in 100 ml buffer) :

● Use: The agarose gel serves as a molecular sieve that separates DNA based on size
during electrophoresis. It is made from seaweed-derived agarose, which forms a porous
network when cooled after being dissolved in a buffer.

· The size of the pores is determined by the concentration of agarose, which

allows smaller DNA fragments to move more easily through the gel than
larger fragments.
 · Why this is important: Different forms of plasmid DNA (e.g., supercoiled,
relaxed, or linear) migrate differently through the gel. Supercoiled plasmid
DNA is compact and moves faster than linearized DNA, while nicked or
relaxed forms move slower. The gel allows you to differentiate between these
forms and assess the quality and purity of the plasmid.

· NOTE: A lower concentration gel is better for separating larger plasmids

(>10 kb), while higher concentrations are used for smaller plasmids and
fragments.

2. TAE Buffer (10 ml of TAE buffer in 490 ml distilled water):

Composition -

1. 40 mM Tris Base

2. 20 mM acetic acid

3. 1mM EDTA

● Use: The buffer provides the necessary ions to carry the electric current through the gel
and maintain a stable pH during electrophoresis.

· TAE (Tris-Acetate-EDTA) is commonly used because it provides a low

ionic strength, meaning it generates less heat during electrophoresis. This
makes TAE ideal for longer runs or when we want to recover DNA from the
gel for cloning or other downstream applications.

○ TBE (Tris-Borate-EDTA) has a higher ionic strength, giving better resolution for
smaller DNA fragments, which is ideal when we need sharper bands, but it is not
suitable for recovering DNA due to its higher salt content.
● Why this is important: The buffer ensures that the DNA molecules remain negatively
charged, which is essential for them to migrate toward the positive electrode. Without a
proper buffer, the DNA may not migrate properly, and the separation may be ineffective.
● Role of Distilled Water: When preparing the buffer from concentrated stock, we dilute
them using distilled water to make a 1x working solution. Distilled water ensures that the
buffer remains free of contaminants like minerals or ions that could alter the ionic
strength or conductivity of the buffer, which would affect DNA migration through the
gel.
3. Loading Dye (1 : 5; Loading Dye : DNA Ratio (2ul) ):

· Use: The loading dye is mixed with our DNA samples before loading them into the
wells. It has two major purposes:

● Density: It contains glycerol or sucrose, which adds weight to the DNA sample so it can
sink into the wells rather than floating away in the buffer.
● Tracking: The dye also contains colored dyes (usually bromophenol blue and sometimes
xylene cyanol or orange G) that migrate through the gel at known speeds, allowing us to
visually track the progress of electrophoresis.

Why this is important:

● Tracking dye helps us gauge when to stop running the gel based on where the dye front
has reached (e.g., bromophenol blue migrates like a 300 bp DNA fragment).
● Without loading dye, we risk the DNA sample not settling in the wells properly, or you
won’t know how far the DNA has migrated, leading to poor separation.

5. Ethidium Bromide (EtBr) (10 ul) :

● Use: Ethidium bromide (EtBr) is an intercalating dye that binds to DNA and fluoresces
under UV light, making the DNA bands visible after electrophoresis.
○ EtBr is commonly used because it’s highly sensitive and binds to both single-
stranded and double-stranded DNA. However, it’s also mutagenic and toxic, so
safer alternatives like SYBR Safe or GelRed are used in some labs.
○ Staining methods:
■ Pre-staining: Adding the dye to the agarose before the gel solidifies, so the
DNA becomes stained as it migrates through the gel.
■ Post-staining: Soaking the gel in an EtBr solution after electrophoresis,
which allows us to stain the DNA bands.
● Why this is important: Without EtBr or an alternative stain, we wouldn’t be able to see
the DNA bands after electrophoresis. The UV transilluminator reveals the fluorescent
bands, allowing us to visualize and photograph the gel for analysis.

6. Plasmid DNA Sample (load 20 ul) :

 ● Use: The plasmid DNA sample is the material we are analyzing. Depending on what we
are looking for, the analysis can confirm:
○ Size of the plasmid: If the plasmid is intact or if it matches the expected size.
○ Plasmid forms: Plasmids can appear in different forms, such as supercoiled,
nicked (relaxed), or linear.
■ Supercoiled DNA is the native form of plasmid DNA, where the strands
are tightly coiled and migrate faster through the gel.
■ Nicked or relaxed plasmid occurs when one strand of the plasmid is
broken, causing it to open up, making it migrate slower.
■ Linear DNA occurs if both strands are cut, which also causes it to migrate
slower than supercoiled DNA but faster than nicked DNA.
● Why this is important: This analysis helps ensure that our plasmid preparation is clean
and intact before proceeding with further experiments. Also, if the plasmid has been
digested by restriction enzymes, the gel will show the expected fragments, confirming the
digestion was successful.

Principle-
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme
digestion., to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to
size fractionate DNA molecules, which then could be purified from the gel if necessary To
separate DNA using agarose gel electrophoresis, the DNA is loaded into precast wells in the gel
and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively
charged, therefore when placed in an electric field, DNA fragments will migrate to the positively
charged anode. different plasmid forms move at varying speeds like Supercoiled DNA (compact,
tightly wound)will migrates faster, open circular DNA (relaxed, less dense) will moves slower,
Linear DNA resulting from enzymatic digestion or damage, migrates in between these two. The
agarose gel forms a porous matrix through which DNA molecules navigate .The gel’s pore size
can be adjusted by varying agarose concentration, which influences the separation efficiency.
Once separation occurs, the DNA is visualized using intercalating dyes like ethidium bromide,
These dyes bind to DNA and fluoresce under UV light, allowing clear visualization of the
plasmid bands.
Protocol-
● Gel Preparation:
1. A 1.2% agarose gel was prepared by weighing the appropriate amount of low
electroendosmosis (EEO) agarose in Tris acetate EDTA (TAE) buffer.
2. The flask was heated using a hot plate until the agarose was completely dissolved. The
agarose was swirled and mixed.
3. The agarose solution was allowed to cool (~55°C) at room temperature. Ethidium bromide
(EtBr) solution was added to a concentration of 0.5 g/ml and swirled. Caution was taken as it is
a carcinogen.
4. The agarose solution was poured onto the assembled gel tray (with barriers in place to hold
the contents of the poured gel). The gel comb was inserted and allowed to polymerize. The
agarose solidified after a few minutes and turned translucent and glossy.

● Sample Loading:
1. The tray with the solidified gel was placed into the electrophoretic box, and the comb was
gently removed from the gel.
2. It was ensured that the wells of the gel were at the black (negative) end of the gel box. DNA,
being negatively charged, runs toward the positive charge (the red side).
3. TAE buffer was poured into the gel box, ensuring that the entire gel was covered and the
wells were filled with buffer.
4. The plasmid DNA sample was prepared by adding 6X DNA loading dye at a 1:5 ratio (loading
dye: DNA).
5. The sample mixture was loaded into the wells of the submerged gel using a micropipette (20
μl of sample was loaded).
 ● Running the gel:
10.The lid of the gel box was placed properly, ensuring that black was aligned with black, and
red with red.
11.The gel was run at a voltage of 1-5 V/cm for about 45 minutes, or until the blue dye had
migrated about 1/2 to 2/3 across the gel.
12.Once the DNA samples or dyes had migrated a sufficient distance, the electric current was
turned off, and the leads and lid were removed from the gel.
13.The agarose gel was visualized and analyzed on a UV transilluminator, and an image was
captured.
Observation and Result-

The solution went whitish after the agar powder was added to the TAE buffer as agar is insoluble
in the TAE buffer. It then became entirely transparent after heating on the hotplate and cooled
before being put into the gel casting tray.
The gel's color did not alter once GelRed was added.
When GelRed is added to the agar gel, it binds to the DNA molecule and makes the DNA bands
visible when the gel is illuminated under a UV light.
The suitable wells for sample loading were created once the gel comb was inserted.
The plasmid DNA sample turned blue after the loading dye was added since it is the color of the
loading dye.
In order to monitor the migration of DNA during gel electrophoresis, loading dye is introduced.
Blue coloured DNA bands can be observed after running the plasmid DNA sample for some-
time.
Red coloured bands of plasmid DNA samples can be observed under UV trans-illuminator.

Figure 1- Plasmid DNA sample + Loading dye.

Figure 2- Agarose Gel Electrophoresis.

Left tube- Plasmid DNA + Loading dye.

Right tube- Plasmid DNA.

Agar gel.
Gel casting tray.

Agarose gel electrophoresis setup.

Red coloured bands of plasmid DNA (Group 1 – 5th well).
Discussion-

Gel electrophoresis is a molecular biology technique used for the separation of DNA, RNA and
protein depending on their molecular size. In this procedure, the plasmid isolated from the E.coli
was run on the Agar gel to determine the presence of DNA by the acquisition of a yellow colour
band. The agar powder & TAE buffer solution turned transparent after heating indicated the
complete dissolution of agar powder as agarose dissolves at 55°C - 100°C. After pouring the gel
and casting it, 8 wells were formed, to which the DNA solution along with the staining dye to
track these DNA bands. GelRed, a safer alternative to ethidium bromide was added to the agar
solution as it binds to the DNA molecule upon illumination under UV which helps us monitor
and visualise the DNA band. GelRed is an intercalating agent that sits between the base pairs of
DNA which is made up of 2 ethidium subunits, bridged by a linear oxygenated space. Depicting
in the observation above, the blue coloured DNA bands are due to the presence of bromophenol
blue and Xylene cyanol that stains the DNA blue, it also indicates the presence of DNA on the
agar gel, due to the process of DNA migration under the presence of an electrical field. This
migration is due to two forces that act simultaneously on the DNA :

1] Frictional force - that prevents the further movement of DNA.

Fe = qF

2] Electrostatic force - which allows the movement of DNA.

Ff = Fu

The DNA gets segregated into different bands representing a specific molecular weight, size and
composition. This separation is based on strength of electrical field, size, conformation of DNA
and density of agarose used. Although we can visualise all fragments of DNA using gel
electrophoresis, smaller fragments or rare fragments may not always be visible owing to low
sensitivity of gel electrophoresis. This issue can be fixed by adjusting the pore size of the agarose
gel. Presenting two scenarios:

1] Higher gel concentration (1.2% - 1.5%) - creates more crosslinks and smaller pore size which
provides apt sensitivity and specificity.

2] Lower gel concentration (0.7% - 1%) - creates fewer crosslinks which permits the large DNA
molecules to migrate further.

The issues of sensitivity can also be corrected by the implementation of southern blotting. In
Southern blotting probes used can detect even low quantities of target DNA, making the method
more sensitive than basic staining techniques used in gel electrophoresis.