Gel electrophorosis final

Gel Electrophoresis

NAYANA.P and Jitendra Kumar
Dept OF FRM
COLLEGE OF FISHERIES

jitenderanduat@gmail.com

Introduction
Gel

electrophoresis is a method used in clinical
chemistry to separate proteins by charge and or
size and in biochemistry and molecular biology to
separate a mixed population
of DNA and RNA fragments by length, to
estimate the size of DNA and RNA fragments or
to separate proteins by charge.
Nucleic acid molecules are separated by applying
an electric field to move the negatively charged
molecules through an agarose matrix.


Gel

electrophoresis separates molecules on
the basis of their charge and size. The
charged macromolecules migrate across a
span of gel because they are placed in an
electrical field.
The gel acts as a sieve to retard the passage
of molecules according to their size and
shape.
Shorter molecules move faster and migrate
farther than longer ones because shorter
molecules migrate more easily through the
pores of the gel. This phenomenon is called
sieving

Gel

electrophoresis is usually performed for
analytical purposes, often after amplification of
DNA via PCR, but may be used as a
preparative technique prior to use of other
methods such as mass spectrometry, RFLP,
PCR, cloning, DNA sequencing, or
Southern blotting for further characterization.


Types of gel
Agarose
Polyacrylamide
Starch


Agarose
 gels

are easily cast and handled compared
to other matrices, because the gel setting is
a physical rather than chemical change.
Samples are also easily recovered. After the
experiment is finished, the resulting gel can
be stored in a plastic bag in a refrigerator.
advantages: it is used for the separation of
DNA fragments ranging from 50 base pair to
several millions of bases using specialized
apparatus. The distance between DNA
bands of a given length is determined by the
percent agarose in the gel

The

disadvantage of higher concentrations is
the long run times (sometimes days).
Low percentage gels are very weak and may
break when you try to lift them. High
percentage gels are often brittle and do not
set evenly
Agarose gels do not have a uniform pore size,
but are optimal for electrophoresis of
proteins that are larger


Polyacrylamide
Polyacrylamide gel

electrophoresis
(PAGE) is used for separating proteins
Pore size is controlled by controlling the
concentrations of acrylamide and bisacrylamide powder used in creating a gel.


Starch
Partially

hydrolyzed potato starch makes
for another non-toxic medium for
protein electrophoresis.
The gels are slightly more opaque than
acrylamide or agarose.
Non-denatured proteins can be
separated according to charge and size.
They are visualized using Napthal Black
or Amido Black staining.

How does it work?
•

DNA is cut into smaller fragments.

•

Loading dye is used to indicate the
fragments of DNA are behind the dye

•

The negative DNA molecule is attracted
to the positive electrode.

•

The smallest fragments move the
greatest distance.

Gel electrophoresis
A

method of separating DNA
in a gelatin-like material using
an electrical field
◦ DNA is negatively charged
◦ when it’s in an electrical field it
moves toward the positive side

DNA

–

→→→→→→→
“swimming through Jello”

+


Gel electrophoresis
DNA

moves in an electrical field…

◦ so how does that help you compare DNA
fragments?
 size of DNA fragment affects how far it travels
 small pieces travel farther
 large pieces travel slower & lag behind
DNA

–

→→→→→→→
“swimming through Jello”

+


Gel Electrophoresis
DNA &
restriction enzyme

longer fragments

wells
power
source

gel
shorter fragments

+

completed gel


fragments of DNA
separate out based
on size

Running a gel

cut DNA with restriction enzymes

1

2

3

Stain DNA
◦ ethidium bromide
binds to DNA
◦ fluoresces under UV
light

Procedure
Remove

comb and observe wells.
Place carbon paper in each end of the tray.
Cover with buffer, making sure the allow
buffer to overflow into each end of the tray.
Load gels.
Connect the electrodes.
Turn on power supply.
Allow gels to run – make sure you see
bubbles coming from the electrodes.

PROCEDURE (CONTINUED)
It

will take about 30 minutes for the gel
to run.
Turn off power supply and remove
electrodes.
Pour off buffer into the designated
container.
Carefully remove gel from gel box and
place in glad container and cover with
stain.
Store in appropriate location.

Applications
Estimation

of the size of DNA molecules
following restriction enzyme digestion, e.g. in
restriction mapping of cloned DNA.
Analysis of PCR products, e.g. in
molecular genetic diagnosis or genetic
fingerprinting
Separation of restricted genomic DNA
Gel electrophoresis is used
in forensics, molecular
biology, genetics, microbiology and biochemist
ry.

Proteins

can also be run on gels. Most
commonly proteins are run on gels made
of polyacrylamide in the presence of SDS
Scientific dyes can also be separated by
gel electrophoresis.


Gel electrophorosis final

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Gel electrophorosis final