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Agarose Gel Electrophoresis
Gel electrophoresis is a widely used technique for theanalysis of nucleic acids and proteins. Agarose gelelectrophoresis is routinely used for the preparation andanalysis of DNA.
Gel electrophoresis is a procedure that separatesmolecules on the basis of their rate of movementthrough a gel under the influence of an electrical field.
We will be using agarose gel electrophoresis todetermine the presence and size of PCR products.PCR products indicate the presence of Wolbachia.
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http://gslc.genetics.utah.edu/units/biotech/gel/
Virtual Gel Electrophoresis
Additional Information onGel Electrophoresis:
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DNA is negatively charged.
+-
Power
DNA
When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
H
O2
An agarose gel is used to slow the movement of DNA and separate by size.
Scanning Electron Micrographof Agarose Gel (11 m)
Polymerized agarose is porous,
allowing for the movement of DNA
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+-
Power
DNA
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel
Size of the DNA!
*Small DNA move faster than large DNAgel electrophoresis separates DNA according to size
smalllarge
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
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Agarose
Agarose is a linear polymer extracted from seaweed.
D-galactose 3,6-anhydroL-galactose
Sweetened agarose gels havebeen eaten in the Far East sincethe 17th century.
Agarose was first used in biologywhen Robert Koch* used it as aculture medium for Tuberculosisbacteria in 1882
*Lina Hesse, technician and illustrator for
a colleague of Koch was the first to
suggest agar for use in culturing bacteria
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Making an Agarose Gel
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An agarose gel is prepared
by combining agarosepowder and a buffersolution.
Agarose
Buffer
Flask for boiling
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Casting tray
Gel combs
Power supply
Gel tank Cover
Electrical leads
Electrophoresis Equipment
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Gel casting tray & combs
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Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching thesurface of the casting tray.
Preparing the Casting Tray
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Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that isseveral times larger than the volume of buffer.
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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 to allow all the grains of agaroseto dissolve.***Be careful when boiling - the agarose solution may become superheated and
may boil violently if it has been heated too long in a microwave oven.
Melting the Agarose
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Allow the agarose solution to cool slightly (~60C) and then carefullypour the melted agarose solution into the casting tray. Avoid air
bubbles.
Pouring the gel
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Each of the gel combs should be submerged in the melted agarose solution.
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When cooled, the agarose polymerizes, forming a flexible gel. It shouldappear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
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Place the gel in the electrophoresis chamber.
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buffer
Add enough electrophoresis buffer to cover the gel to a depth ofat least 1 mm. Make sure each well is filled with buffer.
Cathode
(negative)
Anode (positive)
wells
DNA
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6X Loading Buffer: Bromophenol Blue (for color) Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel, andincreases the density of the samples, causing them to sink into the gelwells.
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Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.The sample should sink into the well. Be careful not to puncture thegel with the pipette tip.
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Place the cover on the electrophoresis chamber, connecting the electricalleads. Connect the electrical leads to the power supply. Be sure the leadsare attached correctly - DNA migrates toward the anode (red). When thepower is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
Running the Gel
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wells Bromophenol Blue
Cathode(-)
Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correctdirection. Bromophenol blue will run in the same direction as the DNA.
DNA(-)
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100
200 300
1,650
1,000
500
850
650
400
12,000 bp
5,000
2,000
DNA Ladder Standard
Inclusion of a DNA ladder (DNAs of know sizes) on the gel
makes it easy to determine the sizes of unknown DNAs.
-
+
DNAmigration
bromophenol blue
Note: bromophenolblue migrates atapproximately thesame rate as a 300bp DNA molecule
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As an alternative to purchasing costly DNA ladders, one can be createdusing meal worm (Tenebrio molitor) DNA and a restriction enzyme.
http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf
St i i th G l
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Staining the Gel
***CAUTION! Ethidium bromide is a powerful mutagen and is
moderately toxic. Gloves should be worn at all times.
Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
Ethidium bromide can be added to the gel and/or running bufferbefore the gel is run or the gel can be stained after it has run.
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Safer alternatives to Ethidium Bromide
Methylene Blue BioRAD - Bio-Safe DNA Stain
Wards - QUIKView DNA Stain
Carolina BLU Stain
others
advantagesInexpensiveLess toxicNo UV light requiredNo hazardous waste disposal
disadvantagesLess sensitiveMore DNA needed on gelLonger staining/destaining time
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Staining the Gel
Place the gel in the staining tray containing warm diluted stain. 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.
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Ethidium Bromide requires an ultraviolet light source to visualize
Vi li i h DNA ( hidi b id )
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Visualizing the DNA (ethidium bromide)
100 200 300
1,650
1,000
500
850 650
400
5,000 bp 2,000
DNA ladder
DNA ladder
PCR Product
1 2 3 4 5 6 7 8
wells
+ - - + - + + -
Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA
Primer dimers
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Visualizing the DNA (QuikVIEW stain)
250
1,500 1,000
500 750
2,000 bp
DNA ladder
PCRProduct
wells
+ - - - - + + - - + - +
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA