Agarose gel electrophoresis by hs

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Agarose Gel ElectrophoresisPresented By-Hari
Shankar VishwakarmaM.Tech (Biochemical
Engineering)harihbti_at_gmail.com
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• Introduction
• Electrophoresis is the migration of charged
  particles or molecules in a medium under the
  influence of applied electric field.
• Electrophoresis was observed first time in 1807
  by Reuss .
• There are several types of electrophoresis, but
  the concepts are similar.
• The electrophoresis machine has an anode
  (positive end) and a cathode (negative end), that
  attract negatively and positively charged
  molecules respectively.

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Principle

• Electrophoresis is a technique, used to separate
  macromolecules especially proteins and nucleic
  acids , that differ in size.
• Negative ions move toward the anode, and
  positive-charged ions move towards the cathode.

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Agarose Gel Electrophoresis

• Agarose gel electrophoresis is a method to
  separate DNA, or RNA molecules by size. This is
  achieved by moving negatively charged nucleic
  acid molecules through an agarose.
• Matrix with an electric field (electrophoresis),
  shorter molecules migrate faster than longer ones
  .

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Making an Agarose Gel
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An agarose gel is prepared by combining agarose
powder and a buffer solution.
Buffer?
Flask for boiling?
Agarose?
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Electrophoresis Equipment
Power supply?
?Cover
Gel tank?
Electrical leads ?
Casting tray?
Gel combs?
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Gel casting tray combs
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Preparing the Casting Tray
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.
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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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Melting the Agarose
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.

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Pouring the gel
Allow the agarose solution to cool slightly
(60ºC) and then carefully pour the melted
agarose solution into the casting tray. Avoid
air bubbles.
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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 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 electrophoresis chamber.
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DNA?
buffer ?
?
?
?
? wells
Anode? (positive)
?Cathode (negative)
Add enough electrophoresis buffer to cover the
gel to a depth of at least 1 mm. Make sure each
well is filled with buffer.
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Sample Preparation
Mix the samples of DNA with the 6X 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.
6X Loading Buffer ? ? Bromophenol Blue (for
color) ? Glycerol (for weight)
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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 the
gel with the pipette tip.
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Running the Gel
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.
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Cathode (-)
? wells
? Bromophenol Blue
DNA (-) ?
Gel
Anode ()
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.
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DNA Ladder Standard
-
Note bromophenol blue migrates at approximately
the same rate as a 300 bp DNA molecule
bromophenol blue?

Inclusion of a DNA ladder (DNAs of know sizes) on
the gel makes it easy to determine the sizes of
unknown DNAs.
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Advantages and Disadvantages of Agarose Gel
Electrophoresis

• Advantages-
• The main benefit of Agarose gel technique is that
  it can be easily processed.
• The DNA molecule that is used as a sample can
  also be recovered without any harm to it at the
  end of process.
• Agarose gel does not denature the DNA sample and
  they stay in own from.
• Disadvantages-
• There is also a disadvantage of gel
  electrophoresis that it may melt (62-65C) when
  electric current is passed through it. Due to
  this reason there are chance that genetic
  material can adopt the shape which are not
  needed.

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Applications of Electrophoresis

• DNA Analysis- Electrophoresis is one way of
  analyzing DNA may be used in forensic
  investigations, DNA sequencing paternity tests
  etc.
• Protein Analysis- Electrophoresis technique is
  used in analysis of structure and function of
  proteins.
• Antibiotics Analysis- With electrophoresis, not
  only able to synthesize new antibiotics but are
  also able to analyze which types of bacteria are
  antibiotic-resistant.
• Vaccine Analysis- Vaccine analysis is one of the
  many important applications of electrophoresis.
  There are several vaccines that have been
  purified, processed and analyzed through
  electrophoresis, such as the influenza vaccine,
  hepatitis vaccine and polio vaccine.

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References

• Wilson Walker, Principle and Techniques of
  Biochemistry and Molecular Biology, Page no.
  449-483.
• Upadhyay Upadhyay, Biophysical Chemistry
  Principles and Techniques, Page no. 422-475.

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