Agarose Gel Electrophoresis

Introduction
 A technique used in Biochemistry and molecular
biology to separate DNA or RNA molecules

 Electrical current is used to create electric field

across the agarose matrix

 The negatively charged nucleic acid molecules

will migrate across the gel matrix towards the
positive electrode

 Migration is based on their molecular size

 Shorter molecules move faster and migrate
further compared to the longer molecules
because they can move through pores of the gel
easily

 Agarose gel electrophoresis will be then stained

with ethidium bromide for DNA visualization

 Discrete bands will be observed under the UV

light
 Agarose is preferred over acrylamide because
it is less toxic and easy to handle although
acrylamide gives better resolution

 Agaropse Gel electrophoresis needs Buffer

solution (TBA/ TAE), Agarose, power supply,
UV lamp to visualize DNA in the gel,
Molecular weight dye such as bromophenol
blue, An ultraviolet-fluorescent dye such as
Ethidium bromide
Components of Agarose Gel
Electophoresis
 Buffer solution
 Agarose
 Ethidium bromide (5.25 mg/ml in H2O)
 Nitrile rubber gloves
 Bromophenol blue
 Glycerol
 Gel rack
 Comb
 UV lightbox
Recipe for Agarose

1. Add 0.5g of agarose into 50mL of TBE Buffer.

2. Microwave to dissolve agarose.
3. Cool the agarose to 60°C.
4. Pour mixture into tank.
5. Place comb.
Principle
 Agarose gel electrophoresis is a widely used method
that separates molecules based upon charge, size
and shape.
 Useful in separating charged biomolecules such as
DNA, RNA and proteins.
 Proteins and nucleic acids are electrophoresed
within an agarose "gel"
  The gel is cast in the shape of a thin slab, with wells
for loading the sample. The gel is immersed within
an electrophoresis buffer that provides ions to carry
a current and some type of buffer to maintain the pH
at a relatively constant value.
 A direct current power supply is connected to
the electrophoresis apparatus and current is
applied.
 When charged molecules are placed in an
electric field, they migrate toward either the
positive or negative pole according to their
charge.
~Molecules having a net negative charge
migrate towards the positive electrode
(anode)
~while net positively charged molecules
migrate towards the negative electrode
(cathode).
~In contrast to proteins, which can have
either a net positive or net negative
charge, nucleic acids have a consistent
negative charge imparted by their
phosphate backbone, and migrate toward
the anode.
 Agarose gels have a large range of
separation, but relatively low resolving
power. By varying the concentration of
agarose, fragments of DNA from about 200
to 50,000 bp can be separated using standard
electrophoretic techniques.
 Inject DNA ladder (molecular weight markers)
into first well

Inject DNA samples into the following wells

DNA ladder and DNA samples are

stained with loading dye (beta-mercaptoethanol)
 Apply current

Small DNA strands move faster and

further through the gel

Stain with Ethidium bromide (EtBr)

Visible under ultraviolet (UV) light

Visualization & Result
Analysis
 Staining
 Ethidium Bromide, visualized under UV light
 Blue excitable stain (eg. SYBR Green), visualized
using blue light excitation source
 Safer
 Passes through transparent plastic and glass
A 1% agarose 'slab' gel prior to UV illumination,
behind a perspex UV shield. Only the marker dyes
can be seen.
The gel with UV illumination, the ethidium
bromide stained DNA glows orange.
Gel stained SYBR® Green I nucleic acid gel
stain.
Digital photo of the gel
after visualized under
UV illumination.
Applications
 The preparation of the agarose gel is a vital step
in determining the well separated DNA bands.
-It able to resolve different sizes of DNA
fragments.
 High concentration (1.5% or 2%) of agarose
- resolves small DNAs (0.2-1.2kb)
 Low concentration (0.7%) of
agarose
- resolves large DNAs (8-10kb)
Applications
 Molecular biology based work:
- to purified the specific sizes of the DNA which are from
the restriction enzyme digestion.
- to obtain the cut plasmids (separating cut vector frm
uncut one)

 Prior to Southern blot transfer

- to separate the genomic DNA frm the RE digested DNA

 Analysis the PCR product

-to target the amplified DNA
 Estimation of the size of the
studied DNA molecule
- by using the DNA ladder
(marker) of various known
band sizes

 Determination of the DNA quantity & DNA quality:

(i) DNA quantity as identified by the λ DNA ladder with
various known DNA in different bands (ruler)

(ii) DNA quality is directly can be observed frm the gel

surface- any absence of streaking or fragment
(contaminated DNA molecule)
Other applications:
 DNA fingerprinting (forensic science)
- to target suspected DNA molecule from criminals

Alkaline agarose gel electrophoresis

- buffer used is substituted by NaOH
- to analyze ssDNA specifically

 Pulse d field electrophoresis

- current flow is periodically altered
- to separate DNA ranging frm 50,000
to 5 million bp