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

More Related Content

What's hot

Viewers also liked

Similar to Gel electrophorosis final

More from College of Fisheries, KVAFSU, Mangalore, Karnataka

Recently uploaded

Gel electrophorosis final