JOMO KENYATTA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF PHARMACY

MASIKA LENNOX

HSP201-0028/2020

PHA2210

MOLECULAR BIOCHEMISTRY

LABORATORY REPORT 2

GEL ELECTROPHORESIS
INTRODUCTION

Gel electrophoresis is a method that separates macromolecules (based on size, electrical charge
and other physical properties) such as nucleic acids or proteins. The electrophoresis term is used
to describe the migration of charged particle under the influence of an electric field. Thus, gel
electrophoresis refers is the technique in which molecules are forced across a span of gel,
motivated by an electrical current. On either end of the gel there are activated electrodes that
provide the driving force. Therefore, a molecule’s properties especially the possession of
ionizable groups, determine how rapidly an electric field can move the molecule through a
gelatinous medium. DNA technology has triggered research advances in almost all fields of
biology. Currently hundreds of useful products are produced by genetic engineering. It has
become routine to combine genes from different sources, usually different species in test tubes,
and then transfer this recombinant DNA into living cells where it can be replicated and
expressed. The most important achievements resulting from recombinant DNA technology
have been advances in our basic understanding of eukaryotic molecular biology. For example,
only through the use of gene-splicing techniques have the details of eukaryotes gene
arrangement and regulation been opened to experimental analysis. Gel Electrophoresis is one of
the staple tools in molecular biology and is of critical value in many aspects of genetic
manipulation and study. One use is the identification of particular DNA molecules by the band
patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral
DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this
way. Another use is the isolation and purification of individual fragments containing interesting
genes, which can be recovered from the gel with full biological activity. Gel electrophoresis
makes it possible to determine the genetic difference and the evolutionary relationship among
species of plants and animals. Using this technology, it is possible to separate and identify
protein molecules that differ by as little as a single amino acid. One very important application
for gel electrophoresis is in DNA Technology. We are now using biotechnology to study the
basic processes of life, diagnose illnesses, and develop new treatments for diseases. Some of the
tools of biotechnology are natural components of cells. For example, Recombinant DNA
sequences contain genes from two or more organisms. This technique has allowed researchers
to gain the ability to diagnose diseases such as sickle cell anemia, cystic fibrosis, and
Huntington's chorea early in the course of the disease. Many researchers are also applying the
techniques of biotechnology to find new treatments for genetic diseases. DNA technology has
triggered research advances in almost all fields of biology. Currently hundreds of useful
products are produced by genetic engineering. It has become routine to combine genes from
different sources, usually different species--in test tubes, and then transfer this recombinant
DNA into living cells where it can be replicated and expressed. Gel Electrophoresis is one of
the staple tools in molecular biology and is of critical value in many aspects of genetic
manipulation and study. One use is the identification of particular DNA molecules by the band
patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral
DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this
way. Another use is the isolation and purification of individual fragments containing interesting
genes, which can be recovered from the gel with full biological activity. Gel electrophoresis
makes it possible to determine the genetic difference and the evolutionary relationship among
species of plants and animals. Using this technology, it is possible to separate and identify
protein molecules that differ by as little as a single amino acid. Gel electrophoresis has two
types:
1) Agarose gel electrophoresis.
2) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Principle:
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to
separate DNA, or RNA molecules by size. This is achieved by moving negatively charged
nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate farther than longer ones.

Principle:
SDS PAGE uses an anionic detergent (SDS) to denature proteins. the protein molecules
become linearized. One SDS molecule binds to 2 amino acids. Due to this, the charge to mass
ratio of all the denatured proteins in the mixture becomes constant. These protein molecules
move in the gel (towards the anode) on the basis of their molecular weights only & are
separated. The charge to mass ratio varies for each protein (in its native or partially denatured
form). Estimation of molecular weight would then be complex. Hence, SDS denaturation is
used.

MATERIAL AND REAGENTS

i. Agarose powder,
ii. 10x TAE buffer,
iii. Microwave
iv. DNA sample,
v. DNA stain (Ethidium bromide)
vi. Loading dye (Bromophenol blue)
vii. Electrophoresis system
viii. Running Buffer (TAE buffer)
ix. DNA ladder

METHODS AND PROCEDURE

Gel electrophoresis.
Set Up Your Gel
Electrophoresis Chamber 1.

Preparation of Agarose Gel

1% agarose gel*
Measured 0.5 g of agarose in a weighing balance
Mixed agarose powder with 50 mL 1xTAE in a microwavable flask/beaker
Microwaved for 3 min until the agarose was completely dissolved (do not over boil the solution
to avoid evaporation of the buffer as this will alter the final percentage of agarose in the gel)
Allowed the solution to cool down to about 50 °C.
Add Ethidium Bromide to a final concentration of approximately 0.5 μg/mL (approximately 1μl
of lab stock solution per 50 mL of gel solution). binds to the DNA and allows visualization of
the DNA under ultraviolet (UV) light.
Poured the agarose into a gel tray with the comb well placed (Any bubbles can be pushed away
with a pipette tip towards the edges of the gel.
Allowed the gel to solidify at 4°C for 10-15 mins.
Placed the casting tray, with the solidified gel in it, onto the central platform in the gel box. The
wells should be at the negative (cathode) end of the box where the black electrical lead is
connected. Very carefully removed the comb from the gel by gently pulling it straight up
Poured about 275 ml of electrophoresis buffer into the electrophoresis chamber. Pour enough
buffer into the box until it just covers the wells of the gel by 1–2 mm.
4. Loaded Samples and Ran them by Electrophoresis
1. Pipet 10 µl from each
tube (1 and 6 DNA ladder, 2, 3, 4 and 5) into separate wells in the gel chamber. Use a fresh tip
for each
tube.
Gels are read from left to right. To keep things straight, the first sample is typically loaded in
the well
at the upper left-hand corner of the gel. For example, Lane 1 2 3 4 5 and 6.
2. Slid the cover of the chamber into place, and connect electrical leads to the power supply,
anode to
anode (red to red) and cathode to cathode (black to black). Make sure both electrical leads are
attached to the same channel of the power supply.
3.Electrophorese at 100 V for 30–40minutes. Shortly after the current is applied, the loading
dye can be
seen moving through the gel
toward the positive side of the gel chamber.
4. When electrophoresis was complete, turned off the
power supply, disconnect the leads from the inputs, and remove the top of gel chamber.
5. Removed the casting tray from gel chamber. The gel is very slippery. Hold the tray level. 6.
Poured the excess buffer back into the original container for reuse, if desired.
Read the results using uv transilluminator

RESULTS AND DISCUSSION

Restriction Digest results
Well 1 and 6 – molecular ladder 1kb
Well 2- Lambda DNA
Well 3- Lambda DNA + Not I restriction enzyme
Well 4 – Lambda DNA + EcorI
Well 5 – Lambda DNA + EcorI + Not I

Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA.

Estimation of the size of DNA molecules

Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern
analysis.
The agarose gel electrophoresis is widely employed to estimate the size of DNA fragments after
digesting with restriction enzymes, e.g. in restriction mapping of cloned DNA.
Agarose gel electrophoresis is commonly used to resolve circular DNA with different
supercoiling topology, and to resolve fragments that differ due to DNA synthesis.
In addition to providing an excellent medium for fragment size analyses, agarose gels allow
purification of DNA fragments. Since purification of DNA fragments size separated in an
agarose gel is necessary for a number of molecular techniques such as cloning, it is vital to be
able to purify fragments of interest from the gel.

Advantages of Agarose Gel Electrophoresis

For most applications, only a single-component agarose is needed and no polymerization
catalysts are required. Therefore, agarose gels are simple and rapid to prepare.
The gel is easily poured, does not denature the samples.
The samples can also be recovered.

Disadvantages of Agarose Gel Electrophoresis

Gels can melt during electrophoresis.
The buffer can become exhausted.
Different forms of genetic material may run in unpredictable forms

REFERENCES
1. https://pdfs.semanticscholar.org/36cf/d4ada922c44d233b6ebfa2af2c956c92e4ec.pdf
2. https://www.mun.ca/biology/scarr/Gel_Electrophoresis.html
3. https://www.wou.edu/las/physci/ch462/Gel%20Electrophoresis.pdf
4. https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
5. https://msu.edu/course/css/451/Lecture/PT-electrophoresis%20(2009).pdf
6. http://library.umac.mo/ebooks/b28050459.pdf