7 steps How to prevent Thalassemia : Dr Sharda Jain & Vandana Gupta
MBAT S2 ABHISHEK GEL ELECTROPHORESIS.pptx
1. SUBJECT : MODERN BIO ANALYTICAL
TECHNIQUES
KARNATAKA COLLEGE OF PHARMACY
TOPIC : GEL ELECTROPHORESIS
SUBMITTED BY: SUBMITTED TO:
ABHISHEK Dr. HARSHA K. TRIPATHY
M. PHARM 2ST SEMESTER ASSOCIATE PROFESSOR
DEPT. OF PHARMACEUTICAL ANALYSIS DEPT. OF PHARMACEUTICAL ANALYSIS
KARNATAKA COLLEGE OF PHARMACY KARNATAKA COLLEGE OF PHARMCY
BANGALURU BANGALURU
1
KARNATAKA COLLEGE OF
PHARMACY
2. DEFINITION
• Gel electrophoresis is a technique used to separate DNA fragments (or other
macromolecules, such as RNA and proteins) based on their size and charge.
• Separated based on their size, charge, molecular weight in the presence of an
electric field using gels as a supporting media.
• Charged molecules move through a gel when an electric current is passed across
it.
• Different kind of gels used are Agar and Agarose gel, starch, sephadex,
polyacrylamide gel.
2
KARNATAKA COLLEGE OF PHARMACY
4. In Agarose gel electrophoresis, agarose is used as a matrix to separate
molecules of varying sizes. it is more commonly used to separate DNA from a
few hundred base pairs or more.
In Polyacrylamide Gel Electrophoresis, polyacrylamide is used as the gel
matrix to separate molecules of varying sizes. It is widely used for the
separation of proteins and DNA fragments with low molecular weight.
KARNATAKA COLLEGE OF PHARMACY 4
5. REQUIREMENTS
1. Electrophoresis apparatus : costing tray of glass or plastic, comb contains
varying number of teeth (for formation of well).
2. Buffer : Carry current and maintain pH of constant value
Ex: Tris Acetate EDTA, Tris Bromate EDTA.
3. Power Supply: The electrodes are connected to their respective terminals
of chamber and power supply with controlled rate of current for best
resolution 5 volts per cm to the gel.
5
KARNATAKA COLLEGE OF PHARMACY
6. 4. Supporting Media(Gel): Starch, Agar/Agarose, cellulose acetate,
polyacrylamide. The choose of supporting media depends on type of
molecule to be separated.
5. Detection techniques:
• Southern blotting (for DNA)
• Northern blotting (for RNA)
• Western blotting (for Protein)
6
KARNATAKA COLLEGE OF PHARMACY
7. PRINCIPLE:
• Based on the principle of migration of charged molecule by application of
electric field and separation takes place based on size, charge, and structure
through a gel matrix.
• This means that a small DNA molecule will travel a greater distance through
the gel than will a larger DNA molecule.
7
KARNATAKA COLLEGE OF PHARMACY
9. INSTRUMENTATION
• Step 1 : preparing the sample for running
• Step 2 : Preparation of gel and buffer
• Step 3 : Load samples
• Step 4 : Electrophoresis
• Step 5 : Visualizing the DNA.
9
KARNATAKA COLLEGE OF PHARMACY
10. 1. PREPARING THE SAMPLE FOR RUNNING
• To begin gel electrophoresis, you will mix your samples with a loading buffer.
• Loading buffer contains both dye, as a visual indicator while loading and
running the sample, and glycerol, to increase the density of the samples.
• Increasing sample density promotes sinking to the bottom of the wells during
loading, preventing the otherwise light samples from quickly diffusing out of
the wells during loading.
10
KARNATAKA COLLEGE OF PHARMACY
11. 2. PREPARATION OF GEL AND BUFFER
During electrophoresis, TAE buffer provides source and ions for setting up the
electric field.
1. 1g agarose + 100 ml TAE = a 1 % Agarose gel solution (Tris Acetate EDTA
buffer).
2. Add Agarose in presence of heat buffer solution poured in tray and cooled.
3. It forms a solid and slightly squinty gel slab with rows of wells at the top
11
KARNATAKA COLLEGE OF PHARMACY
12. 3. LOAD SAMPLES
• Before loading the samples, decide on the ideal order of the samples on the
gel.
• Using a pipette, carefully add samples to individual wells in the gel.
• Additionally, a ladder with specific size markers needs to be added to one of
the wells as a reference for downstream analysis.
12
KARNATAKA COLLEGE OF PHARMACY
13. 4. ELECTROPHORESIS (RUNNING THE GEL)
• Once the samples are loaded, place the lid on the gel box, plug the cords into
the power supply, and run the gel with electrophoresis.
• The voltage and time required will need to be adjusted based on each lab’s
specific experiment.
• Negatively charged sample will start to migrate the gel towards positive.
13
KARNATAKA COLLEGE OF PHARMACY
14. 5. VISUALIZING THE DNA
• Once the dye has migrated from sample, power supply is turned off.
• Gel is removed and placed in ethidium bromide solution.
• For DNA gels, a DNA stain added to the gel allows visualization when
placed under UV light.
• DNA is viable in UV-light.
• DNA strains are visualized from each lane corresponding to chamber well.
• Length of DNA is estimated.
14
KARNATAKA COLLEGE OF PHARMACY
15. APPLICATION
• This method can verify amplification by PCR or sequencing reactions.
• It is used in DNA fingerprinting and the detection of genetic variants and
proteins involved in health and disease as well as in
• It is used for detection and purification of nucleic acids and proteins for
research.
• It is also used to aid in the detection of pathogens (disease-causing organisms)
that may be present in blood or other tissues or in sources such as food.
• Purification of stains cells.
• Used in medical industry.
15
KARNATAKA COLLEGE OF PHARMACY
