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Basics
Speed dependent on their charge, shape,and size, hence electrophoresis has beenextensively developed for molecular
separations.
It is used chiefly for analysis and
purification of very large molecules suchas proteins and nucleic acids,
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Factors AffectingElectrophoresis
1. The Electric Field.
1. Voltage.
b. Current.
c. Resistance.
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Factors AffectingElectrophoresis
2. The Sample
a. Charge.
b. Shape.
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Factors AffectingElectrophoresis
3. The Buffer
a. Composition.
b. Concentration.
c. pH.
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Factors AffectingElectrophoresis
4. The Supporting Medium.
a. Adsorption.
b. Electro – osmosis.
c. Molecular Sieving.
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Gel Electrophoresis.
Separation of high molecular weightsubstances such as proteins andnucleic acids
Improved resolution since the gel iswater insoluble and hydrophilic.
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Gel Electrophoresis.
Eg: Starch, agar and polyacrylamide.
Molecular sieving property of gels helpsto separate large ionic compoundshaving similar charge but different sizeand shape.
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Starch Gel Electrophoresis
Starch gels are prepared by heatingand cooling mixture of partiallyhydrolysed starch resulting in a semi
rigid gel formation. Good for separating complex mixtures
of structural molecules andphysiologically active proteins.
Analysis of isoenzyme patterns(zymograms)
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Agarose Gel
Electrophoresis Agar is a cheap,
non toxic,
chemically illdefined,complex,powdery mixture.
Contains twogalactose basedpolymers,agarose and
agaropectin.
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Agarose GelElectrophoresis
Dissolves in boiling aqueous buffers andforms a gel at 38°C.
Large pore size and low frictional resistance.
Facilitates separation of macromolecules. Suitable for chemical staining after
separation.
Excellent for detection of antigenic proteinsby isotachophoresis andimmunoelectrophoresis.
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DNA separation usingAgarose Gel Electrophoresis
Gel electrophoresis is a widely usedtechnique for the analysis of nucleic acidsand proteins. Agarose gel electrophoresisis routinely used for the preparation andanalysis of DNA.
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Equipment
1. A power pack.
Supplies direct
current betweenthe electrodesbetween theelectrophoresis
unit.
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Equipment
2. ElectrophoresisUnit
a. Electrodes.
b. Buffer reservoirs.
c. Support medium.
d. Transparentinsulating cover
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• DNA is negatively charged.
+-
Powe
r
DNA
• When placed in an electrical field, DNA will migrate toward thepositivepole (anode).
H O
2
• An agarose gel is used to slow the movement of DNA andseparate by size.
Scanning ElectronMicrograph of Agarose Gel
• Polymerized agarose isporous,
allowing for the
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+-
Power
DN
A
How fast will the DNAmigrate?strength of the electrical field, buffer, density of agarose
gel…Size of the DNA!
*Small DNA move faster than large DNA…gel electrophoresis separates DNA according tosize
smalllarge
Within an agarose gel, linear DNA migrate
inversely proportional to the log10 of theirmolecular weight.
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Making an AgaroseGel
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An agarose gel is
prepared by combiningagarose powder and abuffer solution.
Agarose
Buffer
Flask for boiling
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Casting tray
Gel
combs
Power supply
Gel tank Cover
Electrical leads
ElectrophoresisEquipment
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Gel casting tray &
combs
Preparing the Casting
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7/3/12 Seal the edges of the casting tray and put in the combs. Place thecasting tray on a level surface. None of the gel combs should betouching the surface of the casting tray.
Preparing the Casting Tray
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Agarose
Buffer Solution
Combine the agarose powder and buffer solution. Use a flaskthat is several times larger than the volume of buffer.
Melting the
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Agarose is insoluble at room temperature
(left). The agarose solution is boiled until clear
(right).Gently swirl the solution periodically when heating toallow all the grains of agarose to dissolve.
***Be careful when boiling - the agarose solution maybecome su erheated and ma boil violentl if it has
Melting theAgarose
Pouring the
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Allow the agarose solution to cool slightly (~60ºC) and thencarefully pour the melted agarose solution into the casting
tray. Avoid air bubbles.
Pouring thegel
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7/3/12 Each of the gel combs should be submerged in the melted agarose
solution.
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7/3/12 When cooled, the agarose polymerizes, forming a flexible gel.It should appear lighter in color when completely cooled (30-45
minutes). Carefully remove the combs and tape.
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Place the gel in the electrophoresischamber.
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buffer
Add enough electrophoresis buffer to cover the gel to adepth of at least 1 mm. Make sure each well is filled withbuffer.
Cathode(negative)
Anode(positive
)
wells
DNA
S l
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6X Loading Buffer:Bromophenol Blue (for
color)
Glycerol (for weight)
SamplePreparation
Mix the samples of DNA with the 6X sample loading buffer (w/tracking dye). This allows the samples to be seen when loadingonto the gel, and increases the density of the samples, causingthem to sink into the gel wells.
L di th
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Loading theGel
Carefully place the pipette tip over a well and gently expel thesample. The sample should sink into the well. Be careful not topuncture the gel with the pipette tip.
R i th
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Place the cover on the electrophoresis chamber, connecting theelectrical leads. Connect the electrical leads to the power supply.Be sure the leads are attached correctly - DNA migrates towardthe anode (red). When the power is turned on, bubbles should
form on the electrodes in the electrophoresis chamber.
Running theGel
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wells Bromophenol Blue
Cathode(-)
Anod
e(+)
Gel
After the current is applied, make sure the Gel is running in thecorrect direction. Bromophenol blue will run in the samedirection as the DNA.
DNA(-)
DNA Ladder
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100
200
300
1,650
1,000
500
850
650
400
12,000bp
5,000
2,000
DNA LadderStandard
Inclusion of a DNA ladder (DNAs of know sizes) on thegel makes it easy to determine the sizes of unknownDNAs.
-
+
DNAmigratio
n
bromophenolblue
Note:bromophenolblue migratesat
approximatelythe same rateas a 300 bpDNA molecule
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As an alternative to purchasing costly DNA ladders, one can becreated using meal worm (Tenebrio molitor ) DNA and a restrictionenzyme.
http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf
Staining the
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Staining theGel
***CAUTION! Ethidium bromide is a powerful mutagen
and is moderately toxic. Gloves should be worn at
• Ethidium bromide binds to DNA and fluoresces under UVlight, allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or runningbuffer before the gel is run or the gel can be stained after ithas run.
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Safer alternatives to EthidiumBromide
Methylene Blue
BioRAD - Bio-Safe DNA Stain
• Ward’s - QUIKView DNA Stain
• Carolina BLU Stain
…othersadvantagesInexpensiveLess toxicNo UV light requiredNo hazardous wastedisposal
disadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time
St i i th
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Staining theGel
• Place the gel in the staining tray containing warm dilutedstain.• Allow the gel to stain for 25-30 minutes.• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Ethidi B id i lt i l t li ht t
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Ethidium Bromide requires an ultraviolet light source tovisualize
Visualizing the DNA (ethidium
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Visualizing the DNA (ethidiumbromide)
100
200
300
1,650
1,000
500
850
650
400
ß 5,000bp
2,000
DNA ladderDNA ladder
PCR
Product
1 2 3 4 5 6 7 8
wells
+ - - + - + + -
Samples # 1, 4, 6 & 7 were positive for WolbachiaDNA
Primerdimers
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Visualizing the DNA (QuikVIEW stain)
250
1,500
1,000
500
750
2,000bp
DNA ladder
PCRProdu
ct
wells
+ - - - - + + - - + -
+
March 12, 20
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia
DNA
l l id l
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Polyacrylamide GelElectrophoresis.
Polyacrylamide gels are physicallytougher than agarose gels.
The gel forms when a mixed solution of
acrylamide and cross-linker monomersco-polymerize into long chains that arecovalently cross-linked.
The most common cross-linker is N,N'-methylenebisacrylamide (“bis”).
P l l id G l
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Polyacrylamide GelElectrophoresis.
Porosity of gel is determined by relativeproportion of acrylamide monomer tocross linking agent
Gels may be defined in terms of total %of acrylamide present.
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SDS PAGE
Sodium dodecylsulphate polyacrylamidegel electrophoresis.
Principle: SDS is an anionic detergent
which binds to proteins causing theirdenaturation.
In excess of SDS the proteins have aconstant negative charge.
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SDS PAGE
Protein-SDS complex moves towardsanode.
Mobility inversely proportional to log of
molecular weight. Molecular weights of unknown proteins
can be determined.
P l l id G l
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Polyacrylamide GelElectrophoresis.
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Polyacrylamide Gel
Electrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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y yElectrophoresis.
Polyacrylamide Gel
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y yElectrophoresis.
Polyacrylamide Gel
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y yElectrophoresis.
Polyacrylamide Gel
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y yElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.
Polyacrylamide Gel
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Polyacrylamide GelElectrophoresis.