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 quicly molecules will tra!el
through the gel, and thus what their migration
distance will be.
Small size !s. "arge size
Strong charge !s. wea charge
Introduction
Short repetiti!e interspersed Elements #SINE$
%onsists of relati!ely short sequences #&' to a few
hundred base pairs in length$
E(ist at numerous places throughout the genome
The Alu sequence
Introduction
The Alu Sequence
)'' base pair sequence that e(ist at more than
*'',''' places throughout the human genome
+unction not completely understood
Doesnt code for any type of protein or DNA
P,-. "ocus on %hromosome &/
homozygous or heterozygous
Ob0ecti!es
to separate and fractionate #using Agarose Gel
Electrophoresis$ the isolated DNA in the
pre!ious e(periment
to be able to draw conclusions based on
different sizes and charges that migrate
through a gel during electrophoresis
to determine whether the sub0ect is
homozygous or heterozygous for the alu gene
PRO%ED1RE
Assembly of Agarose Gel
Electrophoretic %ell
Agarose Gel Preparation
&2 agarose in &''m" of &( TAE buffer
Solubilized in heat
%ool down to *' 3 /' %
Place the comb near the edge
Pour gel o!er the chamber
Pour &( TAE buffer into the gel tan
Remo!e combs
"oading of DNA samples
Lane Sample Load Volume
& 44R #DNA standard$ &' ul
. 5omozygous #676$ %ontrol &' ul
) 5omozygous #878$ %ontrol &' ul
9 5eterozygous #678$ %ontrol &' ul
* Student & .' ul
/ Student . .' ul
: Student ) .' ul
; Student 9 .' ul
Agarose Gel Electrophoresis
DNA Separation
Positi!e electric charge is applied to the negati!ely
charged nucleic acid molecules
Shorter molecules mo!e faster and migrate
farther than longer ones
4igration affected by factors such as pore size,
!oltage used and length of DNA sample
4ET5ODS
D. Staining
&. Stain gels for .8) min with &''( +ast <last
DNA Stain
4ET5ODS
D. Staining
.. Rinse gels for &' seconds with *''8:''m"
of clean, warm #9'8**=%$ tap water.
4ET5ODS
D. Staining
). >ash with *''8:''m" of clean, warm tap
water twice for *min each.
4ET5ODS
D. Staining
9. E(amine for DNA bands #fuzzy at first$
4ET5ODS
E. ,isualization of DNA
Gel Documentation System
Results and Discussion
Alu
Small, repetiti!e DNA elements of around )'' bp
repeated almost *'','''( throughout the human
genome
Dimorphic 3 insertion may be present or absent on
each of the paired chromosomes of different people
PV92 region of chromosome &/
/9& bp intron
)'' bp insertion #Alu$
Results
Lane Sample
& 44R
. #676$ control
) #878$ control
9 #678$ control
* Student & #chee$
/ Student . #chee$
: Student ) #hair$
; Student 9 #hair$
& . ) 9 * / : ;
Lane Sample Base Pairs Genotype
& DNA Standard
.
5omozygous #676$
%ontrol
-9&
5omozygous #676$
)
5omozygous #878$
%ontrol
/9&
5omozygous #878$
9
5eterozygous #678$
%ontrol
-9& and /9&
5eterozygous #678$
* Student & -9& and /9& 5eterozygous #678$
/ Student . -9& 5omozygous #676$
: Student ) -9& 5omozygous #676$
; Student 9 -9& and /9& 5eterozygous #678$
Guide ?uestions
If ethidiumbromide were to be used
as DNA !isualizing agent, what
precautions would be obser!ed
when handling this reagent@ >hy@
Ethidium<romide
),;8Diamino8*8ethyl8/8
phenylphenanthridinium
bromide
intercalating agent
Ethidium<romide
Red cationic fluorescent
dye
,isualize DNAs and
RNAs in electrophoresis
gels
+luoresces readily into
an orange7 reddish8
brown color when
e(posed to 1, light
Ethidium<romide is a 4utagen
4ay cause genetic damage
4oderately to(ic after an acute e(posure
%an be absorbed through the sin
Irritant to sin, eyes, mouth and 1RT
Safety +irstA
Substitute.
>or in a suitable en!ironment.
Place in shatter8proof, lea proof container
when transporting.
4aintain a clean setting.
1se PPEs.
Emergency e(posure procedures
Dispose properly.
Personal Protecti!e Equipments
Protecti!e clothing
As much co!erage as possible
Eye protection
Safety glasses with side shields
%hemical splash goggles
Glo!es
Disposable nitrile glo!es, change frequently
5andwash thoroughly after
Emergency E(posure Procedure
I44EDIATE medical attention
Eyes 3 irrigate for &* mins
Sin 3 wash with soap and water
Swallowed7inhaled 3 medical attention
Proper Disposal
All contaminated articles must be treated as
hazardous waste and labeled as such.
>hen using a 1, transilluminator to
!isualize DNA what safety
precautions should be obser!ed and
why@
1, Transilluminator
Emit relati!ely high
le!els of 1, radiation
Effects of 1, Radiation
Photoeratitis
Ocular cataracts
Erythema
<listering
Sin aging
Sin cancer
Safety +irstA
Substitute.
PPEs #mas, gown, nitrile glo!es$
Shielding
>arning signs