gel electrophoresis to separate DNA RNA

1. Agarose Gel
Electrophoresis
PRESENTED BY:-
AVINASH SHARMA
PALB-3235
power
supply
gel box
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2. Contents:-
Definition of agarose gel electophoresis
Procedure of agarose gel electrophoresis
Application of gel electrophoresis
Problem in agarose gel electrophoresis
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3. What is agarose?
• it is a polymer of D,L galactose and it is a heteropolysaccharide extracted
from red algae seaweed.
• Low electro endosmosis and good for working.
• Agarose has large range of separation but low resolving power.
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4. Agarose Gel Electrophoresis
Gel electrophoresis is a procedure that separates molecules on the basis of
size and/or charge and/or shape under the influence of an electrical field.
It is based on the principle of “ohms law equation” and”power equation”.
Gel electrophoresis is a widely used technique for the separation of
nucleic acids and proteins. Agarose gel electrophoresis is routinely used
for the separation of DNA.
e will be using agarose gel electrophoresis to
determine the presence and size of PCR
products. +-
Power
DNA
H
O2
+
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5. Procedures of agarose gel electrophoresis:-
An agarose gel is prepared by
combining agarose powder
and a buffer solution.
Agarose
Buffer
Flask for boiling
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6. Casting tray
Gel combs
Power supply
Gel tank Cover
Electrical leads

Electrophoresis Equipment
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7. Gel casting tray & combs
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8. 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 the surface of the casting
tray.
Preparing the Casting Tray
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9. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is several
times larger than the volume of buffer.
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10. 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 agarose to
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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11. Allow the agarose solution to cool slightly (~60ºC) and then carefully pour
the melted agarose solution into the casting tray. Avoid air bubbles.
Pouring the gel
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12. Each of the gel combs should be submerged in the melted agarose solution.
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13. When cooled, the agarose polymerizes, forming a flexible gel. It should appear
lighter in color when completely cooled (30-45 minutes). Carefully remove the
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14. Place the gel in the electrophoresis chamber.
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15. buffer 
Add enough electrophoresis buffer to cover the gel to a depth of at least
1 mm. Make sure each well is filled with buffer.
Cathode
(negative)
Anode
(positive)

wells
  
DNA
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16. Loading Buffer: 
• Bromophenol Blue (for color)
• Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with the sample loading buffer ( tracking dye). This
allows the samples to be seen when loading onto the gel, and increases the
density of the samples, causing them to sink into the gel wells.
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17. 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 the gel with the
pipette tip.
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18. Place the cover on the electrophoresis chamber, connecting the electrical leads.
Connect the electrical leads to the power supply. Be sure the leads are attached
correctly - DNA migrates toward the anode (red). When the power is turned on,
bubbles should form on the electrodes in the electrophoresis chamber.
Running the Gel
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19.  wells
 Bromophenol Blue
Cathode
(-)
Anode
(+)
Gel
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA
(-)

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20.  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.
-
+
DNA
migration
bromophenol blue
Note: bromophenol blue
migrates at
approximately the same
rate as a 300 bp DNA
molecule
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21. Staining the Gel
***CAUTION! Ethidium bromide is a powerful mutagen and is
moderately toxic. Gloves should be worn at all times.
• Ethidium bromide fluorescent dye and binds to the DNA under UV light
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel .
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22. 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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23. Ethidium Bromide requires an ultraviolet light source to visualize
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24. Factors affecting the DNA in gel
elctrophoresis:-
• Agarose concentration:-
↑ conc. Of agarose then migration larger fragments of dna and vice versa so for
larger fragments of dna agarose conc.=0.7% and smaller fragments of dna
agarose conc.=1.5%.
• Voltage:-
Voltage increases then migration of larger fragments of dna will be more than
the smaller dna.so voltage should not be more than 5volts/cm.
• Electrophoresis buffer:-
TAE and TBE buffers are used in the gel electrophoresis because it establish
the pH8.0 and provide ions for the migration of dna and ionic strength are
different.
• Effects of ethidium bromide:-
It is a carcinogenic mutagen and fluorescent dye which are used in the
visualizing the dna when agarose gel are placed in the UV transilluminator
then dye are free in the gel then dye binds with dna then it produce
orange light for visualizing the dna.
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25. Application of electrophoresis
• To separate fragments from each other
• To determine the sizes of fragments
• To determine the presence or amount of DNA
• To analyze restriction digestion products prior to southern transfer or
of RNA prior to northern transfer.
• Analysis of PCR product.
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26. Proplems in agarose gel electrophoresis:-
• Smearing
•voltage too high for large fragments
•too much DNA
• Gel melts
•voltage too high
•ionic strength too low
• Poor resolution
•wrong [agarose]
•small bands are fuzzy – the gel run may have been too long at too low
a voltage, allowing diffusion of the DNA and broadening of the band
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Thank you for your attention