agarose gel electrophoresis
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Transcript of agarose gel electrophoresis
Introduction
What is Gel Electrophoresis?
It is a basic biotechnology technique that separates macromolecules according to their size and charge
There are two types of gel electrophoresis namely Polyacrylamide gel and Agarose gel electrophoresis
What is its application?
It is frequently used to analyze and manipulate samples of DNA, RNA, or proteins.
Introduction
What to expect?
• Size and net charge are factors that together
determine how quickly molecules will travel
through the gel, and thus what their migration
distance will be.
– Small size vs. Large size
– Strong charge vs. weak charge
Introduction
• Short repetitive interspersed Elements (SINE)
– Consists of relatively short sequences (10 to a few
hundred base pairs in length)
– Exist at numerous places throughout the genome
– The Alu sequence
Introduction
• The Alu Sequence
– 300 base pair sequence that exist at more than
500,000 places throughout the human genome
– Function not completely understood
• Doesn’t code for any type of protein or DNA
• PV92 Locus on Chromosome 16
– homozygous or heterozygous
Objectives
• to separate and fractionate (using Agarose Gel
Electrophoresis) the isolated DNA in the
previous experiment
• to be able to draw conclusions based on
different sizes and charges that migrate
through a gel during electrophoresis
• to determine whether the subject is
homozygous or heterozygous for the alu gene
• 1% agarose in 100mL of 1x TAE buffer
• Solubilized in heat
• Cool down to 50 – 60 C
• Place the comb near the edge
• Pour gel over the chamber
• Pour 1x TAE buffer into the gel tank
• Remove combs
Lane Sample Load Volume
1 MMR (DNA standard) 10 ul
2 Homozygous (+/+) Control 10 ul
3 Homozygous (-/-) Control 10 ul
4 Heterozygous (+/-) Control 10 ul
5 Student 1 20 ul
6 Student 2 20 ul
7 Student 3 20 ul
8 Student 4 20 ul
Agarose Gel Electrophoresis
• DNA Separation
– Positive electric charge is applied to the negatively
charged nucleic acid molecules
– Shorter molecules move faster and migrate
farther than longer ones
– Migration affected by factors such as pore size,
voltage used and length of DNA sample
Alu
• Small, repetitive DNA elements of around 300 bp
repeated almost 500,000x throughout the human
genome
• Dimorphic – insertion may be present or absent on
each of the paired chromosomes of different people
• PV92 region of chromosome 16
– 641 bp intron
– 300 bp insertion (Alu)
Results
Lane Sample
1 MMR
2 (+/+) control
3 (-/-) control
4 (+/-) control
5 Student 1 (cheek)
6 Student 2 (cheek)
7 Student 3 (hair)
8 Student 4 (hair)
1 2 3 4 5 6 7 8
Lane Sample Base Pairs Genotype
1 DNA Standard
2Homozygous (+/+)
Control941
Homozygous (+/+)
3Homozygous (-/-)
Control641
Homozygous (-/-)
4Heterozygous (+/-)
Control941 and 641
Heterozygous (+/-)
5 Student 1 941 and 641 Heterozygous (+/-)
6 Student 2 941 Homozygous (+/+)
7 Student 3 941 Homozygous (+/+)
8 Student 4 941 and 641 Heterozygous (+/-)
If ethidium bromide were to be used
as DNA visualizing agent, what
precautions would be observed
when handling this reagent? Why?
Ethidium Bromide
• Red cationic fluorescent
dye
• Visualize DNAs and
RNAs in electrophoresis
gels
• Fluoresces readily into
an orange/ reddish-
brown color when
exposed to UV light
Ethidium Bromide is a Mutagen
• May cause genetic damage
• Moderately toxic after an acute exposure
• Can be absorbed through the skin
• Irritant to skin, eyes, mouth and URT
Safety First!
• Substitute.
• Work in a suitable environment.
• Place in shatter-proof, leak proof container when transporting.
• Maintain a clean setting.
• Use PPEs.
• Emergency exposure procedures
• Dispose properly.
Personal Protective Equipments
• Protective clothing
– As much coverage as possible
• Eye protection
– Safety glasses with side shields
– Chemical splash goggles
• Gloves
– Disposable nitrile gloves, change frequently
– Handwash thoroughly after
Emergency Exposure Procedure
• IMMEDIATE medical attention
• Eyes – irrigate for 15 mins
• Skin – wash with soap and water
• Swallowed/inhaled – medical attention
When using a UV transilluminator to
visualize DNA what safety
precautions should be observed and
why?
Effects of UV Radiation
• Photokeratitis
• Ocular cataracts
• Erythema
• Blistering
• Skin aging
• Skin cancer