AGAROSE GEL

ELECTROPHORESIS

Bajalla, Barrameda, De Guzman D.,

Gandola, Ong, Ramos, Rey, San Luis
4C-Biochemistry
Outline
- What is Agarose Gel Electrophoresis?
- Principles Involved in AGE
- Instrumentation/Materials
- Applications
- Studies using Agarose Gel Electrophoresis
Gel Electrophoresis
● Gel electrophoresis is a technique used to separate DNA, RNA and protein
fragments based on their size and charge.

● Electrophoresis involves
an electric current running
through a gel containing the
molecules of interest.

● Gel: agarose gel or

polyacrylamide gel
Agarose Gel Electrophoresis
● Agarose is a linear polysaccharide ( average molecular mass abou 12,000)
made up of basic repeat unit agarobiose, which comprises alternating units
of galactose and 3,6-anhydrogalactose.

● Gel is a matrix of polymers forming

sub-microscopic pores
Agarose Gel Electrophoresis
● Agarose gel has large range of separation but low resolving power.
Amt of Agarose in Gel (%) Efficient Range of Separation of Linear
DNA molecules (kb)

0.3 60-5

0.6 20-1

0.7 10-0.8

0.9 7-0.5

1.2 6-0.4

1.5 4-0.2

2.0 3-0.1
Principles Involved in AGE
● Electric current is used to drive the
movement of the molecules through the
gel
● The molecules being sorted are
dispensed into the well in the gel material;
the gel is then placed in an
electrophoresis chamber
● negatively-charged DNA migrates
(electrophorese) towards the bottom
(cathodal, positive) end
Principles Involved in AGE

FACTORS AFFECTING MIGRATION:

1. Molecular size of DNA

2. Agarose concentration
3. Conformation of DNA
4. Applied current
Materials Needed in Agarose Gel ELectrophoresis
Agarose Gel
● Dry, powdered flakes.
● Dissolved in buffer (TAE or TBE)
● Inert matrix
● Agarose Gels
○ 0.7% gel (large DNA fragments; 5-10kb)
○ 2% gel (small fragments; 0.2-1kb)
Materials Needed in Agarose Gel ELectrophoresis
Casting trays
● Available in many sizes
● Composed of UV-transparent plastic
● Open ends must be closed and sealed

Comb
● Placed in the gel after poured
● Produces wells after removing the comb
Materials Needed in Agarose Gel ELectrophoresis
Buffer System
● Maintains pH
● Generate ions consistently
● TAE (Tris Acetate EDTA) - pH 8.0
○ Resolves high molecular weight
fragments.
● TBE (Tris Borate EDTA) - pH 8.0
○ Resolves low molecular weight
fragments.
Materials Needed in Agarose Gel ELectrophoresis
DNA Ladder
● Standard reference that contains DNA
fragments of known lengths to determine
the size of the unknown DNA.
● Comes in different ranges.
Materials Needed in Agarose Gel ELectrophoresis
Sample preparation
● Samples (DNA) must be mixed with 6x
loading buffer
● Weighs the sample down and allows
visualization of loading
Materials Needed in Agarose Gel ELectrophoresis
Voltage
● Helps the sample to move through the gel.
● The higher the voltage, the faster the DNA will
travel through the gel.
○ Too high voltage
■ Can possibly melt the gel
■ Cause smearing
■ Distortion of DNA bands.
○ Too low voltage
■ Broadening of band for small DNA fragments
(dispersion and diffusion)
Visualization
Ethidium Bromide
● Intercalates into between stacked bases
and commonly used as a fluorescent tag
(nucleic acid stain).
● Fluoresces under UV light (reddish -
orange color)
● UV absorbance (300 - 360)
● Toxic and carcinogenic
● Insert itself between base pairs of the
double helix
● 0.5 - 1 µg/mL (std conc)
Visualization
● Alternatives
○ Methylene Blue
○ Orange G
○ Xylene cyanol
○ Bromophenol Blue
Electrophoresis Equipment
 Method for Electrophoresis
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly

Pour into casting tray and allow to solidify

Add running buffer, load samples with marker

Run the gel at constant voltage until separation occurs

View DNA on UV-light and show results

Applications
● Separation of restriction enzyme digested DNA including genomic DNA
● Analysis of PCR products after polymerase chain reaction to assess for target
DNA amplification
● Allows for estimation of the size of DNA molecules using DNA marker or
ladder which contains DNA fragments of various known sizes
● Allows the rough estimation of DNA quantity and quality
● Criminal cases
● Paternity cases
● Diagnose genetic diseases
● Genetic kinship among species
 It is generally known that the shift of DNA fragments in
agarose gel was primarily depended on the nucleobase
amount of DNA fragment and the voltage of electrophoresis.
The short DNA fragments shift faster than the long fragments,
and the shift rate of DNA fragments is promoted by increasing
the voltage of electrophoresis. The influences of GO on the
shift of DNA fragments in agarose gel were investigated by
adjusting the concentration of GO in agarose gel.
Summary

❏ DNA fragments were adsorbed onto the surfaces of GO

nanosheets dispersed in agarose gel net by
intramolecular interaction.
❏ DNA fragments were desorbed from the surfaces of GO
nanosheets under electrophoresis condition, which could
be influenced by the charges carried on both the DNA
fragments and GO nanosheets.
Summary
❏ The successive absorption-desorption processes between
DNa fragments and GO nanosheets significantly improved
the separation resolution of DNa fragments by increasing
the shift distances between the adjacent DNA fragments
with different nucleobase amounts.
 1
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QUALITY OF DNA EXTRACTED FROM MILK POWDERS
QUALITY OF DNA EXTRACTED FROM MILK POWDERS
SUMMARY
High-quality DNA is relatively difficult to extract from milk powder compared
with raw milk, because milk powder is associated with high-temperature
treatments during processing.

Studies proved that heat treatment has an effect on the quality of DNA
extracted from food material, as it could shorten the length of both mitochondrial
and nuclear DNA fragments.

However, we found DNA extracted from milk powder that experienced a

high-temperature treatment to have a good yield, purity, and integrity. Thus, the
DNA extraction method we described is suitable for milk powder.