Experiment III

Aim: Analysis of DNA by Agarose Gel

Electrophoresis

Dr Pratibha Chanana
Analysis of DNA by Agarose Gel Electrophoresis.

• Separate mixed population of macromolecules such as DNA or

proteins in a matrix of agarose
DNA and RNA fragments are separated by length
Proteins may be separated by charge and/or size
• Biomolecules are separated by applying an electric field to move the
charged molecules through an agarose matrix
• Biomolecules are separated by size in the agarose gel matrix.
• Horizontal gel electrophoresis -
• Use agarose in a submerged horizontal orientation.
 • Principle of separation for all electrophoresis
• Movement of a charged molecule in a medium subjected to an electric
field.
• v=Eq/f

V - velocity of the molecule subjected to electrophoresis.

E - electrical field in volts/cm
q - net charge on the molecule
f - frictional coefficient.
• Impact of f depends on the mass/size and shape of the molecule.
• Counteracting force generated by the viscous drag
Think of a short linear oligonucleotide vs a large supercoiled plasmid vs long
chromosomal DNA.
• Media through which the molecules migrate – Adds a value to f
 Agarose
• Linear polysaccharide (average relative molecular mass
~12,000)
• Made up of basic repeat unit Agarobiose
• Alternating units of Galactose and 3,6 -
anhydrogalactose.
• Agarose Gel –
• 3D matrix formed of helical agarose molecules in Repeating units of Agarose
supercoiled bundles
• Contains microscopic pores that act as a molecular
sieve
• Once hydrated and formed into a gel, carbohydrate
will form helical fibers and aggregates creating
channels of 50 to more than 200 nm in diameter.
• DNA and RNA molecules migrate by 'reptation'(snaking
through this matrix).

Source: University of San Diego HomePage

 Mobility (µ) of molecule in gel is related to agarose concentration (ι):
log µ = log µ0 – Krι
µ0 – free mobility of DNA in solution
Kr – retardation coefficient (relates to properties of gel and migrating molecules)
ι – agarose concentration
Distance migrated in gel decreases as log of molecular weight
Higher percent of agarose in a gel, the more dense agarose and smaller pores in the solidified gel
Types of agarose
• Standard agarose - High strength and high melting temperature.
• Melting temp: 85 - 95◦C
• Gelling tem: 34 - 42◦C
• Low melting temperature Agarose –
• Melts at 63-65◦C and gel at 25-35◦C.
• A quality agarose will have a low electroendo-osmosis (EEO)
 Electro endosmosis (EEO)
• Motion of liquid induced by an applied potential across a porous material,
capillary tube, membrane, microchannel, or any other fluid conduit.
• Caused by stationary charged groups in the gel or along the inner wall of
the vessel.
• Negatively charged acidic groups in the gel or silicate groups embedded in
glass surfaces bind mobile positively charged counterions electrostatically.
• Positively charged counter-ions migrate in the electric field, transporting
their water of hydration
• Giving rise to water flow of EEO in that direction.
• Since the flow rate decreases with increasing distance from the glass wall
• Backbone of the DNA contains a negative charge
for every nucleobase present
• When an electrical field applied to a solution containing DNA
 DNA molecules will migrate towards the positively charged
electrode
 DNA and RNA have a constant charge to mass ratio
(above 400 bp), separation is based on size and shape,
not charge.
 Gels are made by heating up agarose in an appropriate buffer.
 Gel contains microscopic pores that act as a molecular sieve.
Process of Agarose gel electrophoresis
Divided in to four parts:
1) Casting of the gel
2) Loading of the sample
3) Electrophoresis
4) Staining and visualization
Casting
• Video link - https://www.youtube.com/watch?v=dSFQHt-zIQs
 Staining and Visualization
• DNA intercalating agent commonly used as
a fluorescent tag (nucleic acid stain)
• When exposed to ultraviolet light 300nm
• Will fluoresce with an orange colour, intensifying
almost 20-fold after binding to DNA.
• Ethidium bromide(0.5 mg cm -3) commonly used
during DNA fragment separation by agarose gel
electrophoresis.
• Added to buffer and binds by intercalating between
DNA base pairs.
• When the agarose gel is illuminated using UV light,
DNA bands become visible.
DNA loading buffer (6X)

• 30% (v/v) glycerol

Makes the solution dense and allows it to sink to the bottom of the well.
• 0.25% (w/v) bromophenol blue –
Makes it easier to see the sample that is being loaded and also acts as a
marker of the electrophoresis front
• 0.25% (w/v) xylene cyanol FF
Tracking dye
Materials Required

Reagents:
• SRL Biolit Genomic DNA extraction Kit/ Plasmid isolation Kit
• Agarose
• TAE Buffer (Tris/Acetate/EDTA)
• 6X Sample Loading Dye
• Ethidium Bromide Stain
• 6x Sample loading Dye Preparation: 3ml glycerol (30%), 25mg
bromophenol blue (0.25%) dH2O to 10mL.
• TAE Buffer: 40mM Tris, 20mM Acetate and 1mM EDTA; pH ~ 8.6.
• Apparatus - Agarose gel electrophoresis apparatus (Genei).
Procedure:
Preparation of 1% Agarose gel
1) Weigh 200 mg of agarose and place it in a conical flask.
2) Add 20 ml of 1X TAE (from the 50X stock make 1X TAE) to agarose in a conical flask.
3) Boil it in a microwave/ hot plate till the agarose dissolves entirely and the solution looks clear.
4) Allow the hot agarose to cool down and add 3.5µl of Ethidium Bromide and mix by swirling.
5) Finally pour the gel onto a gel plate sealed on either side and insert the comb.
6) Place the casted gel in to the apparatus and fill with running buffer.
Loading of sample
Mix 10µl of Sample + 2µl of 6X loading Dye.
9) Load 12µl of sample into one well of 1% agarose gel and electrophorese.
10) Load 12µl of control DNA in another well
11) Electrophorese at 50V for 30 min or till the dye front reaches the bottom.
12) Observe the bands under UV light or in a Gel Doc
Question
• Stock = 50 X of TAE
• Working solution - 1x TAE - 20 ml
• How much volume of 50 x TAE should we take
 tracks from a 0.8% agarose
submarine gel
OBSERVATION

Genomic DNA

Plasmid DNA-
Supercoiled conformation
Precautions:
• Ethidium bromide is thought to act as a mutagen because it
intercalates double-stranded DNA (i.e. inserts itself between the
strands), deforming the DNA.
• Use gloves while handling and discard the used tips/Gel exposed to
EtBr only in the designated area.
• While viewing, use proper UV shield or use a Gel doc.
• Make sure electrodes are connected properly and in the proper
orientation.