Agarose gel electrophoresis power point presentation on agarose gel electrophoresis

1. GEL
ELECTROPHORESIS
Presented by-Manisha
Phamaceutical chemistry

2. INTRODUCTION
 Gel Electrophoresis is a technique in which we separates the ion or molecules
on the basis of size,shape and total charge.
 The word electrophoresis means-ion carried by electricity in an electric
field.Here the electricity act as a driving force .
 And in electric field the more highly charged ion and ion of smaller size
migrate at faster rate than ions of larger size or lower charge.
 Negatively charged ion like DNA move from CATHODE TO ANODE.
Because CATHODE is a negatively charged electrode and ANODE is positively
charged electrode.
 Positively charged ion move from ANODE to CATHODE.

3. PURPOSE OF GEL ELECTROPHORESIS
A method for
separating DNA
Can be used to
separate the
size of
DNA
RNA
PROTEIN
• We will be using it
to purify DNA,RNA
and PROTEINS.

4. PRINCIPLE OF GEL ELECTROPHORESIS
 Gel Electrophoresis is a type of ELECTROPHORESIS. In which the
Agarose gel is used as a support medium that’s why it is also called
AGAROSE GEL ELECTROPHORESIS
 IN AGAROSE GEL ELECTROPHORESIS DNA is a separating substance .
 Classification of ELECTROPHORESIS is based on their support medium.
AGAROSE GEL ELECTROPHORESIS
SDS PAGE(SOD.DODECYL SULPHATE POLY ACRYLAMIDE
GEL ELECTROPHORESIS)
NATIVE GEL(WITHOUT SDS)
ELECTROFOCUSING GELS

5. MOVEMENT
OF IONS IN
ELECTRIC
FIELD
Positively charged particle move from ANODE
TO CATHODE.
AND negatively charged particles or ions move
from CATHODE TO ANODE.
Size and shape of the particle decide the
velocity with which the particle will migrate
under the given electrical field and the
medium.

6. PROCEDURE OF GEL
ELECTROPHORESIS
REQUIREMENTS
•SAMPLE
•BUFFER SOLUTION
•THE DRIVING
FORCE(ELECTRICAL SUPPLY)
•SUPPORT MEDIUM
•STANDARD(LADDER DNA)
•DETECTING SYSTEM

7. GEL
PREPARATION
 First we need to make
our gel.DNA
ELECTROPHORETIC GEL
(AGAROSE) is extracted
from sea weed.
 Used to separate
macromolecules such as
Nucleic acids,large
proteins and protein
complexes.
 It is prepared by
dissolving 0.5% agarose
in boiling water and
allowing it to cool to 40
degree Celsius.

8. Agarose gel slab preparation:
specified amount of agarose powder is weighed and is dissolved in
boiling water or added to a buffer solution and boiled to form a transparent
solution.
After some cooling this transparent solution is poured in a casting tray (which has
comb) in such way that air bubbles should not be there.
This casting tray is then kept a side at room temp. for 15-20- min. to solidify the
gel.
After gel formation comb is removed from gel. Sample wells are created due to
comb.
Now this solidified agarose gel slab with sample well is removed from casting tray
and used in electrophoresis equipment.
During the boiling followed by cooling process rearrangement of hydrogen bond
takes place and interchain cross linking occurs due to which gel becomes porous.

9. GEL ADDED TO CASTING TRAY

10. It is most widely used polysaccharide gel .
Agarose gives a more uniform degree of
porosity than starch.
Agarose is used in 1% or 2% gel.
Larger pore size is produced by less
concentration of agarose and smaller pore
size is produced by more concentration of
agarose.
Separation is based on charge to mass
ratio of sample particles.
It is mainly used for separation of DNA
fragments.

11. Advantages of agarose gel :
easy to prepare the gel .
Resolution is better than starch gel and paper.
Disadvantage of gel:
purity of agarose is major issue here. If it has more on
sulphate group then agarose is less pure.

12. SAMPLE
PREPARATION
SAMPLE(DNA fragments of different size)
Sample with loading dye and glycerol:
Loading dye move faster than the
segment.Once the dye reaches to the
terminal we get indication that DNA
fragment must of reach somewhere near
and does the power supply has to be
turned off.
Glycerol: it increases the density of
sample so that the sample will layer at
the bottom of a gel’s sample well.

13. BUFFER
 The buffer in electrophoresis has twofold
purpose:
• Carry applied electrical current
• They set the pH as which electrophoresis
is carried out.
 Thus they determine:
• Types on charge on solute.
• Extent of ionization of solute.
• Electrode towards which the solute will
migrate.

14. Importance of EDTA in buffer solution
 It is a chelating agent which chelates magnesium ion .
 Magnesium is the cofactor of DNA nucleases.
 Hence activity of DNA nucleases that may be present is inhibited , and DNA is
protecting from degrading by DNA nucleases.

15. POWER SUPPLY
Drives the movement of ionic
species in the medium and
allow the adjustment and
control of the current or
voltage.
Constant delivery is required.

17. STANDARD
 DNA STANDARD OR LADDER
DNA
 With help of this we can
determine the size of sample
DNA.

20. DETECTING
SYSTEM
 The EtBr(ethidium bromide)
works as a color agent that
gives color to DNA.
 EtBr works as a separating
agent in AGAROSE GEL
ELECTROPHORESIS.
 EtBr intercalates between DNA
base pairs and emits
fluorescence under UV light.
 By using a orange color
filter,the orange color DNA can
be seen.

22. Advantages and disadvantages of gel
electrophoresis
 Advantages
 Gel provides molecular sieve like structure and causes
separation based on molecular size.
 Gel slabs can be prepared by verticle , horizontal
electrophoresis. GE in tubes is also done.
 The gel is mechanically stable. Due to which post
electrophoretic work is possible such as blotting , electro-
elution.

23.  Disadvantages
* it is not a precise technique – it is a semi
quantitative type of technique. To get exact mass of
proteins to one has to go for mass spectroscopy.
* used for analysis of specific sample (such as nucleic acid
,proteins i.e. macromolecules) small hormones
neurotransmitters , and ions can not be measured by
electrophoresis.
*speed of analysis and data production is slow.

24. Application of gel electrophoresis
 Gel electrophoresis is a widely used technique in molecular biology and
biochemistry labs.
 Agaorse GE is widely used in separation of DNA molecules.
 Polyacrylamide gels are mainly used in protein separation and analysis.
 GE is used in forensic analysis for separation of DNA fragments for DNA
fingerprinting to investigate crime scenes.
 GE is used in DNA profiling.
 It is used to analyze PCR products.
 It is used to study of structure and function of proteins.
 Used for antibiotic analysis.

25. APPLICATION OF
ELECTROPHORESIS
1. DNA analysis
2. Protein analysis:throught electrophoresis the
amount of protein in blood or urine is measured
and compared to established normal
value,lower or higher than the normal levels
usually indicates a disease.
3. Serum protein analysis
4. Lipoprotein analysis
5. Small molecules (drugs,steroids) monitoring
6. Urine analysis
7. Cerebrospinal fluid analysis

26. REFERENCES
 Anees A Siddique , Seemi Siddique (2014) pharmaceutical analysis vol.1 , 3rd
ed. Page no. 236-241 CBS publishers and distributors.
 Online resources
 www. Reseachgate.net
https://www.researchgate.net/post/What-is-the-role-of-TAE-in-Gel-
Electrophoresis#:~:text=EDTA%20is%20a%20chelating%20agent,ions%2C%20in%20pa
rticular%20magnesium%20ions.&text=The%20combination%20of%20the%20buffer,f
ragments%20when%20you%20use%20acetate
 Wikipedia.com.
https://en.m.wikipedia.org/wiki/Agarose_gel_electrophoresis#:~:text=For%20a%
20standard%20agarose%20gel,used%20for%20a%20standard%20electrophoresis