This document discusses agarose gel electrophoresis, which is used to separate DNA fragments by size. It begins by explaining the basic principle, which is that charged DNA fragments will move through an agarose gel under the influence of an electric field, with smaller fragments moving faster. It then describes how agarose gel is prepared by mixing agarose with buffer and casting it. Different types of gels, like agarose and polyacrylamide, can be used depending on the size of DNA fragments. The document outlines the steps for running samples on a gel, detecting DNA bands with ethidium bromide staining, and some applications like capillary and pulsed-field gel electrophoresis.

1. Prepared By:
M. Kaleem Iqbal
Roll No: 309
M.Phil Biotechnology 2012-14

2. Principle
 Got its name from electromotive force. Charged
particles move under the influence of electric field
from one electrode to the other.
 If they are passed through a medium they can be
separated on the basis of their sizes.
 DNA is negatively charged, its fragments moves
towards anode.

3. Agarose gel is a polymer
meshwork
++++
POSIT
IVE
ELECT
RODE
------
NEGA
TIVE
ELECT
RODE
Power source
Sample well with
DNA
Movement of DNA
General scheme of Agarose Gel Electrophoresis
Small DNA moves faster Large DNA moves slow

4. Types of Gels used for Electrophoresis
 Depending on the size of fragments different materials
can be used for electrophoresis.
 Agarose: Chains of branched and unbranched
carbohydrates. Mostly used for larger DNA fragments.
Contains no cross links.
 Polyacrylamide: Amides of acrylic acid. Used for
resolving smaller fragments. It forms cross links with
bisacrylamide.

5. Agarose Gel Electrophoresis
 Agarose in solid form is mixed with buffer and heated
for sometime.
 Fix the combs according to required wells in the
casting apparatus.
 Poured in gel casters and allowed to solidify at room
temperature.

6. Polyacrylamide Gel
 Acryamide and bisacrylamide are dissolved in buffer.
 Ammonium persulfate and TEMED are used to
polymerize the gel.
 Gel is poured in casters (plates), fixed the combs and
allowed to stay for sometime to polymerize.

7. Sample preparation
 DNA samples to be run can be generated by the action
of restriction enzymes (as in RFLPs) or it may be a PCR
product.
 Add adequate amount of loading dye. It increases the
density of samples and help in visualization while the
gel is running.

8. Detection
 Ethidium Bromide is added
into the gel prior to
solidification.
 Gel is visualized under UV.
 EtBr. Intercalates with DNA
and gives fluorescence
under UV.

9. Capillary Gel Electrophoresis
 Capillary filled with matrix is used instead of large gel
tank.
 A source vial (anode) and a destination vial (cathode)
both filled with a buffer and a sample vial.
 Capillary is first dipped in sample vial and then in
source vial.
 Movement under osmo-electric flow.

11. Pulsed-Field Gel Electrophoresis
 Specialized to separate larger fragments ranging from
10Kb to 10Mb.
 Current is not passed in one direction, instead
alternatively at different angles and it is not
continuous but in the form of pulses.

12. Schematic diagram of pulse field gel
electrophoresis

13. Applications
 Diseases.
 Sequencing.
 Family.
 Animals.
 Forensics.

14. Thanks