The document discusses agarose gel electrophoresis. It begins with an introduction to electrophoresis and gel electrophoresis, explaining how molecules are separated based on size and charge through an applied electric field in a gel matrix. It then describes the basic components and process of agarose gel electrophoresis, including preparing the agarose gel, loading and running the samples, and visualizing the results to separate DNA fragments. Agarose gel electrophoresis is used to separate nucleic acids like DNA and RNA by size and analyze results like PCR products and DNA molecules.

1. AGROSE GEL ELECTROPHORESIS
Presented by:
Shobhini Chandel
M.Pharm
1stsemester
Submitted to:
Dr. Saumya Das
Associate Professor
NIET(Pharmacy
Institute)

2.  Introduction
 Principle
 Types of gel electrophoresis
 Requirement and Procedure (Agarose gel
electrophoresis)
 References

3.  Electrophoresis is a general term that describes the migration and
separation of charged particles (ions) under the influence of an electric
field.
 Gel electrophoresis is a type of electrophoresis that separates
molecules or components based on their size or conformation in a
matrix made from gel-forming substances under applied electric field.
 In laboratory gel electrophoresis is used to separate mixtures of DNA,
RNA, or proteins according to molecular size.

4.  An electrophoretic system consists of two electrodes of opposite
charge (anode, cathode), connected by a conducting medium called
an electrolyte. The separation effect on the ionic particles results
from differences in their velocity (v), which is the product of the
particle's mobility (m) and the field strength (E):
v= m E
 Charge — higher the charge greater the electrophoretic mobility.
Size — bigger the molecule greater are the frictional and
electrostatic forces exerted on it by the medium. Consequently,
larger particles have smaller electrophoretic mobility compared to
smaller particles.
Shape — rounded contours elicit lesser frictional and electrostatic
retardation compared to sharp contours. Therefore globular protein
move faster than fibrous protein.

5.  Agarose Gel Electrophoresis
 Polyacrylamide Gel Electrophoresis
 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
(SDS PAGE)
 Starch Gel Electrophoresis
 Two -Dimensional Gel Electrophoresis

6.  Agarose is a natural linear polysaccharide isolated from red
seaweed agar.
 In Agarose gel electrophoresis, DNA or RNA molecules can be
separated based on their size. This is achieved by the movement of
negatively charged nucleic acid molecules through an agarose
matrix in a horizontal electrophoresis.
 Molecules with smaller size move faster and migrate farther as
compared to longer ones.
 Agarose gel electrophoresis is generally suitable for the separation
of DNA fragments ranging from 100 base pairs to 20 kb pairs.
https://laborimpex.be/principles-and-
practice-of-agarose-gel-electrophoresis-
manufacturer-reference-369.html

7.  Buffers and Solutions:
Agarose solutions
Ethidium bromide (staining dye)
Electrophoresis buffer (TAE , TBE)
Loading buffer (glycerol and loading dye)
 Nucleic Acids and Oligonucleotides:
DNA samples.
DNA Ladders.
 Equipment and supplies:
An electrophoresis chamber and power supply.
Gel casting trays
Sample comb
Transilluminator

8. https://www.smacgigworld.com/assets/blog-img/Steps-in-
Electrophoresis.webp

9. 1. Prepare a 50x stock solution of TAE buffer in 1000ml of distilled water.
2. Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the
electrophoresis tank and to cast the gel.
3. Prepare a solution of agarose in electrophoresis buffer at an appropriate
concentration ( 0.5%, 0.7%, 1%. 1.2% etc depends upon DNA size)
4. Heat the slurry in a boiling-water bath or a microwave oven until the agarose
dissolves. The agarose solution can boil over very easily so keep checking it.
It is good to stop it after 45 seconds and give it a swirl. Use insulated gloves
or tongs to transfer the flask/bottle into a water bath at 55°C.
5. When the molten gel has cooled, add 0.5µg/ml of Ethidium bromide. Mix
the gel solution thoroughly by gentle swirling.
6. While the agarose solution is cooling, choose an appropriate comb for
forming the sample slots in the gel.
7. Pour the warm agarose solution into the mold.
8. Allow the gel to set completely (30-45 minutes at room temperature), then
pour a small amount of electrophoresis buffer on the top of the gel, and
carefully remove the comb. Pour off the electrophoresis buffer. Mount the
gel in the electrophoresis tank.

10. 9. Add just enough electrophoresis buffers to cover the gel to a depth
of approx. 1mm.
10. Mix the samples of DNA with 0.20 volumes of the desired 6x gel-
loading buffer.
11. Slowly load the sample mixture into the slots of the submerged
gel using a disposable micropipette or an automatic micropipettor.
Load size standards into slots on both the right and left sides of
the gel.
12. Close the lid of the gel tank and attach the electrical leads so that
the DNA will migrate toward the positive anode. Apply a voltage
of 1-5 V/cm.
13. Run the gel until the bromophenol blue and xylenecyanol FF have
migrated an appropriate distance through the gel.
14. The gel tray may be removed and placed directly on a
transilluminator. When the UV is switched on we can see orange
bands of DNA. Comparing these band with DNA ladder we will
get the desired DNA.

11.  Separation of nucleic acid.
 Separation of protein.
 Estimation of the size of DNA molecules
 Analysis of PCR products
 Agarose gel system combined with sodium dodecyl sulfate (SDS)
has been developed to separate large proteins.
 Agarose gel electrophoresis enables you to distinguish DNA
fragments of different lengths.
 Also used in the blotting techniques for analysis of macro
molecules.
https://geneticeducation.co.in/part-2-analyzing-and-
interpreting-agarose-gel-electrophoresis-results/

12.  Fritsch R.J. , Krause I. Encyclopedia of Food Science and Nutrition
(Second Edition) 2003 ,Pg no. 2055
 https://conductscience.com/gel-electrophoresis/ 09/18/2020
 Sankar S.R. Text book of Pharmaceutical Analysis (Third Edition)
Pg no. 28.1-28.8
 https://www.brainkart.com/article/Electrophoresis--Principle-and-
Types_34123/
 https://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2