A molecular biology technique

1. GEL ELECTROPHORESIS
ASSSIGNMENT OF GENETIC ENGINEERING
PAPER CODE= BTI-608
NAME:- RONAK B.LASHKARI
M.SC BIOTECH
IAR NO:=12694

2. Introduction :-
 Electrophoresis come from the Greek word where ‘Electro’ means charged
particle and ‘phoresis ’ means movement.
 DEFINATION :- Electrophoresis is migration of charged particle or a molecule
in a medium under the influence of an applied (external) electric field.
 Electrophoresis is used in laboratories to separate macromolecules based on
size.
 Many biological molecules such as protein ,amino acid, peptides ,nucleotides
,nucleic acid(DNA and RNA) possess ionizable group and therefore at any given
pH ,exists in solution as electrically charged species.
 Under the influence of external electric field , these charged particle will
migrate either to cathode or anode , depending on the nature of their net
charge.
 GEL ELECTROPHORESIS :-
 Gel electrophoresis involve the use of gel as supporting media for separation
of macromolecule( DNA ,RNA ,protein)under the influence of electric charge.

3. Principle :-
 The electromotive force (EMF) generated across the electrode pushes or pulls the molecule
(nucleic acid and protein )through the gel matrix.
 The molecule move towards anode if negatively charged or towards cathode if positively
charged.
 When a potential difference is applied across the electrode of electrophoretic tank
containing ‘agarose gel’ and biomolecules (DNA) are loaded then they get separated
according to their molecular size and move to their respective electrode.[ DNA contain
phosphate group and it is negatively charged so it move towards anode].
 Here the agarose gel act as a sieve .As in the sieve large and bulky particle stay above and
the particle which are smaller than pore size passes through it . Similarly in the gel large
and bulky molecules stay behind and moves slowly whereas the smaller molecules move
faster and quickly towards their respective electrode(anode).
 This result in separation of molecule by size, with the larger molecules near the well and
smaller molecules further away.

4. Requirements:-
1) Electrophoresis chamber and power supply
2) Gel casting trays:- available in variety of size and composed of UV transparent plastic .
The open end of trays are closed with tape while the gel is being cast , then removed
prior to electrophoresis.
3) Sample combs:- around which molten medium is poured to form sample wells in the
gel.
4) Electrophoresis Buffer :- usually, TAE (Tris Acetate EDTA Buffer) and TBE ( Tris Borate
EDTA Buffer)
5) Loading Buffer:- It contains glycerol which increase the weight of the sample and
avoids the oozing out of sample from the well and a tracking dye which gives visibility
to the colourless sample and help in tracking the run.
6) Staining :- DNA molecules are easily visualized under an ultraviolet lamp when
electrophoresed in the presence of extrinsic flour ethidium bromide. Alternatively
,nucleic acids can be stained after electrophoretic separation by soaking the gel in a
solution of ethidium bromide . When intercalated into double stranded DNA
fluorescence of this molecule increase greatly.
7) Transilluminator :- this is an ultra violate box which is used visualize ethidium bromide
Stained DNA into the gel.

5. Steps of gel electrophoresis
1) An Agarose TAE gel solution is prepared :- First of all agarose powder is mixed with elec-
trophoresis buffer to the desired concentration, then heated in a microwave oven until completely
melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 mg/ml) at
this point to facilitate visualization of DNA after electrophoresis.
2) Casting the gel :-After cooling the solution to about 60°C, it is poured into a casting tray containing
a sample comb and allowed to solidify at room temperature. After the gel has solidified, the comb is
removed, carefully.
3) Setting up the electrophoresis chamber :-The gel, still in its plastic tray, is inserted horizontally into
the electrophoresis chamber and just covered with buffer .The gel is positioned so that the chamber
wells are closest to the negative electrode of the chamber.
4) Loading the gel:- Samples containing DNA mixed with loading buffer are then pipetted into the
sample wells and a DNA ladder is also loaded as a reference for size, the lid and power leads are
placed on the apparatus.
5) Electrophoresis :- The negative and positive leads are connected to the chamber and current is
applied. Turning on the power supply sets up the electric field and then negatively charged DNA
sample will migrate towards the positive electrode . The migration of DNA fragment can be
visualized by staining with ethidium bromide.

6. .

7. 6) Stopping Electrophoresis and visualizing the DNA :-
once the DNA sample has migrated through the gel far enough , the power supply is
turned off .Ethidium Bromide interact between the bases of DNA and is visible in UV
light.(but the gel can also be stained after electrophoresis by soaking in a dilute solution of
ethidium bromide). The ethidium bromide stained gel is then exposed to UV light and picture
is taken. The DNA bands are visualized on gel. The DNA ladder that was loaded is also
visualized and length of DNA band can be estimated.

8. Application :-
 Separation of DNA,RNA and Protein .
 Analysis of PCR product.
 In blotting technique for analysis of macromolecule.
 In the separation of DNA fragment for DNA fingerprinting.
 Separation of amino acid and enzymes.
 Separation of antibiotic drug.
 To analyze gene associated with a particular illness .
 It may be used as a preparative technique prior to use of other method such as mass
spectroscopy , DNA sequencing ,cloning ,southern blotting and RFLP.
 It is used in forensic ,molecular biology, genetic engineering, microbiology and
biochemistry.
 In study of evolutionary relationship by analyzing genetic similarity among population
or species.
 In DNA profiling for taxonomy studies to distinguish different species.