Agaro

se Gel

Electrophor
esis

Atar Derj
EGR 3335 Introduction to Biotechnology
Engineering and Management Sciences
Al Akhawayn University

What is Electrophoresis?
Electrophoresis is a laboratory
technique for separating mixtures
of charged molecules.

Mixture: a material composed

of two or more elements or
parts.
Charged Molecules: a molecule
(such as a protein or DNA) that
has too many or too few
electrons.

What does gel electrophoresis do?

Employs electromotive force to move
molecules through a porous gel
Separates molecules from each other
on the basis of
size and/or
charge and/or
shape

Basis of separation depends on how

the sample and gel are prepared

Why perform electrophoresis on ds

DNA?
To separate fragments from each
other
To determine the sizes of fragments
To determine the presence or amount
of DNA
To analyze restriction digestion
products

What do we start with?

What we start with: A variety of
different fragments of DNA all mixed
together
We will use gel electrophoresis to
separate/sort these fragments

What is Agarose?
Linear carbohydrate polymer extracted
from seaweed , agarbiose

Forms a porous matrix as it gels

Shifts from random coil in solution to structure
in which chains are bundled into double helices

Components of an
Electrophoresis System
Power supply and chamber, a
source of power supply
Buffer, a fluid mixture of water and
ions
Agarose gel, a porous material that
molecules migrates through
Gel casting materials

Electrophoresis Equipments
Power supply

Cover
Gel tank

Electrical leads

Casting tray
Gel combs

Cathode

Anode

+
Buffer
Dyes

Power Supply

Separation by Size
As DNA is moved
through the gel,
smaller sized
fragments move
through faster than
larger sized fragments

start

end

Separation Using Charge

The charge on DNA is
what makes it move
through the gel
DNA is a charged
molecule. What is the
charge on DNA?
Negative charge

Why?
Phosphate group is
negatively charged

Separation Using Charge

The gel is hooked up to a
power source
DNA is loaded into the gel on
the cathode (-) end
Gel is placed in a buffer
solution that will conduct
electricity
Electric current is run
through the gel
DNA is attracted to the +
end (anode) = runs to the
red

The Gel
Wells are created to put the DNA
into
We use agarose
gels to separate
wells
DNA
well
-

Direction DNA travels

Direction
DNA
travels

SIDE VIEW

TOP VIEW

Challenges
DNA is colorless-- how will we see
it on the gel & when we are
loading it into the gel?
How do we get the DNA to stay in
the well (not float away)?

Solution #1
Problem #1: How can we see the DNA
sample as we load it into the gel
Problem #2: How can we make sure DNA
wont float away
Solution: Add loading dye to the initial
DNA sample!

Loading Dye
Adds mass to the DNA sample so that it
will go into the well
makes it sink to the bottom

Adds blue color so you can see what you

are pipetting

Solution #2
Problem: DNA is colorless. Once the DNA
has been run through the gel, how can we
see where it is on the gel?
Solution: Add Ethidium Bromide (EtBr) or
Gel Red to the gel

Ethidium Bromide
The DNA intercalates with
the Ethidium Bromide
(EtBr)
Intercalates = inserts itself
between bases

GelRed also stains nucleic

acids
EtBr and GelRed will
fluoresce under UV light

Relative Size vs. Absolute Size

Looking at a gel, you can
determine which fragments
of DNA are bigger than
others = Relative Size
Which fragment is bigger, A
or B?

(-) start

Fragment A (didnt travel as far

in a fixed amount of time)
(+) end

Absolute Size
How can we determine the actual size of the
DNA fragments (how many base pairs- bp)?
Use a size standard
Also called a DNA ladder
Consists of a series of fragments of known sizes
Use it to compare to your DNA fragments

Illustration of Gel
Electrophoresis

- - Negative
Electrode - -

- - Negative Electrode -

Wells

+ + Positive Electrode +
+

Before
Electrophoresis

+ + Positive Electrode +
+

After
Electrophoresis

 Based this info, how

big is the circled
fragment?
850 bp

Sa
mp dard
le
Sa
1
mp
le
2

1000 bp
850 bp
750 bp
600 bp
200 bp
100 bp

Si
ze

Suppose you have a

size standard with
the following sized
fragments: 1000 bp,
850 bp, 750 bp, 600
bp, 200 bp, 100 bp

St
an

Example

Gel Electrophoresis steps

Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.

Pour into casting tray with comb and allow to solidify

Add running buffer, load samples and marker

Run gel at constant voltage until band separation occurs

View DNA on UV light box and document results

Animation
http://l
earn.gen
etics.ut
ah.edu/c
ontent/l
abs/gel/

Real Life Examples of Uses for

Electrophoresis

Law Enforcement
Agencies
Hospitals
Genetics Research

Thank you for your attention