Gel Electrophoresis
Gel Electrophoresis
‫الترحيل الكھربائي عن‬
‫الھالمم‬
‫ريق ھ‬
‫طريق‬
‫إعداد‬
‫ مرتضى عبد المھدي المظفر‬.‫د‬
Molecular virology
E‐ mail : [email protected]
٢
Dr. Murtadha Al‐muthafer
11/5/2014
ELECTROPHORESIS



Electrophoresis is a separation technique that is
based on the movement of charged particles in an
electric field.
field
The term electrophoresis was coined from a
Greek word “Phoresis” which means “Being
Carried Away
Away”..
Hence literal meaning of the word electrophoresis
means “to carry with electricity.”
Why electrophoresis?
Why electrophoresis?
To separate DNA fragments from each other
To determine the sizes of DNA fragments
To determine the
To determine the presence or amount of DNA
To analyze restriction di ti
digestion products
d t
PRINCIPLE
•
•
Any charged ion or molecule migrates when
placed in an electric field, the rate of migration
depend upon its net charge,
charge size
size, shape and the
applied electric current.
Can be represented by following eq .
E*q
q
V
f
FACTORS AFFECTING
ELECTROPHORETIC MOBILITY
1 Charge
1Ch
– higher
hi h the
th charge
h
greater
t the
th
electrophoretic mobility.
2- Size – bigger the molecule greater are the
frictional
ct o a and
a d eelectrostatic
ect ostat c forces
o ces eexerted
e ted oon itt
by the medium. Consequently, larger particles
have smaller electrophoretic mobility
co a ed to smaller
compared
alle particles.
a ticle
3 Shape – rounded contours elicit lesser frictional
3and electrostatic retardation compared to
sharp
p contours. Therefore g
globular p
protein
move faster than fibrous protein.
Gel Electrophoresis
l l
h
Agarose Gel electrophoresis
Strach Gel Electrophoresis
Poly acrylamide
l
l
d
Gel Electrophoresis
Agarose Gel Electrophoresis
• Agarose gel electrophorresis
A
l l t h
i is a method i
th d
to separate DNA or RNA molecules by size. • This is achieved by moving negatively charged nucleic acid molecules through an agarose
nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
• Shorter molecules move faster and migrate g
faster than longer ones .
Instruments and Material required for agarose
gel electrophoresis
gel electrophoresis
 Electrophoresis unit
( Gel chamber, Gel casting tray, A comb )
 Power supply with cathode and anode P
l ih h d
d
d
electrode  Agarose gel
 Buffer
 Staining agent (dye)
 DNA ladder  Sample to be separate
S
l t b
t
Electrophoresis Equipment
Electrophoresis Equipment
What is Agarose ?
• Agarose is a linear polymer extracted from seaweed , l
l
df
d
agarobiose, the agarose gel is used to separate DNA and RNA fragments, types of agarose
d
f
f
;
• Standard Agarose ‐ LE
G l
Gels at 35‐38
3 38oC; Melts at 90‐95
C
l
90 9 oC
Becomes opaque at high concentrations
• Low Melting Agarose
Low Melting Agarose (NuSieve)
Gels at 35oC; Melts at 65oC
Often used to isolate DNA fragments from gel
Often used to isolate DNA fragments from gel
Staining of DNA
g
• To make DNA fragments visible after electrophoresis the DNA must be stained
electrophoresis, the DNA must be stained
• The favorite—ethidium bromide
• When bound to DNA it fluoresces under ultraviolet light (reddish –orange colour)
g (
g
)
• Convenient because it can be added directly to the gel
• Sensitive—detects 0.1µg of DNA
• Othe
Oth alternatives for ethidium
lt
ti
f
thidi
b
bromide :
id
1‐ Methylene blue 2‐ Syber safe
DNA ladder
DNA ladder
• IIt is a solution of DNA i
l i
f DNA
molecules of different length
• DNA Ladder consists of known DNA L dd
i
f k
DNA sizes used to determine the size of an unknown DNA
the size of an unknown DNA sample. • The DNA ladder usually The DNA ladder usually
contains regularly spaced sized samples which when run on an
samples which when run on an agarose gel looks like a "ladder".
Sample preparation
• DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive pole (anode).
• An agarose gel is used to slow the movement of DNA and separate • An agarose
gel is used to slow the movement of DNA and separate
by size. H
O
2


DNA
‐
Power
+
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small
Small DNA move faster than large DNA
DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA
small
large
‐
Power
+
Procedure
Visualization
• After the electrophoresis is complete, the molecules l l in i the th gel l can be b stained t i d to t make k
them visible. photograph h
h can be b taken k
of f the h gel l under d
ultraviolet lighting conditions. If the molecules to be
to be separated
separated contain
contain radioactivity
radioactivity added
added for visibility, an autoradiogram can be recorded of the gel By
recorded of the gel . By
• Transilluminator (UV radiation, 312 nm)
• Instruments for gel documentation (Polaroid camera or a video recorder)
camera or a video recorder) Other instrument
Other instrument • Capillary electrophoresis
• Bioanalyzer
• Tape station • Pulsed field
electrophoresis electrophoresis
• NGS