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GEL ELECTROPHORSIS
Sanjay patil
M- PHARMA [QA]
SOPS RGPV
ELETOPHORSIS
A technique for separating the compound of a
mixture of charged molecular (proteins , DNA
,RNA) in an electr...
GEL ELECTROPHORESIS
 It is a technique used for the separation of
Deoxyribonucleic acid, Ribonucleic acid or protein
mole...
Charged molecules are separated based on their
electrical charge and size.
T YPE OF GEL
Agarose Gel
 A highly purified uncharged polysaccharide
derived from agar.
 Used to separate macromolecule...
POLYACRYLAMIDE GEL
Commonly used components: Acrylamide
monomers, Ammonium persulphate,
Tetramethylenediamine
 These fre...
MATERIAL REQUIRED FOR GEL
ELECTROPHORESIS









Electrophoresis chamber
gel
Gel casting tray
Buffer
Staining ag...
GEL ELECTROPHORESIS EQUIPMENT
GEL COSTING TRAYS

 available in a variety of
sizes and composed of
UV-transparent plastic.
 The open ends of the
trays ...
APPLIED VOLTAGE
 voltage, rate of migration
The higher the voltage, the more quickly the
gel runs
But if voltage is too...
BUFFERS
During electrophoresis water undergoes
hydrolysis : H 2 O  H + OHBuffers prevent the pH from changing by
reacti...
BUFFERS
Another compound is added to make Tris an
effective buffer — either boric or acetic acid
Another compound is add...
ETHIDIUM BROMIDE
 The standard concentration
used in staining DNA in
gels is 0.5-1ug/mL
 Ethidium bromide is a
fluoresce...
STAINING OF DNA
 UV absorbance maxima at 300 and 360 nm and
emission maxima at 590 nm.
 Detection limit of bound DNA is ...
A COMB
 A comb is placed in the
liquid agarose after it
has been poured
 Removing the comb
from the hardened gel
produce...
METHOD FOR ELECTROPHORESIS
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix
thoroughly
Pour into casting tray ...
APPLICATION
Electrophoresis is employed in biochemical
and clinical field.
In the study of protein mixtures
Antigen ant...
Analysis of PCR products, e.g. in molecular
genetic diagnosis or genetic fingerprinting
Separation of organic acid, alka...
THANK YOU
Sanjay
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  1. 1. GEL ELECTROPHORSIS Sanjay patil M- PHARMA [QA] SOPS RGPV
  2. 2. ELETOPHORSIS A technique for separating the compound of a mixture of charged molecular (proteins , DNA ,RNA) in an electric field within a gel or other support. The movement of electrically charged molecular is an electric field often resulting in their separation
  3. 3. GEL ELECTROPHORESIS  It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or protein molecules according to their size and electrical charge using an electric current applied to a gel matrix. What is a gel? Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules.
  4. 4. Charged molecules are separated based on their electrical charge and size.
  5. 5. T YPE OF GEL Agarose Gel  A highly purified uncharged polysaccharide derived from agar.  Used to separate macromolecules such as nucleic acids, large proteins and protein complexes.  It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40 C.
  6. 6. POLYACRYLAMIDE GEL Commonly used components: Acrylamide monomers, Ammonium persulphate, Tetramethylenediamine  These free radicals activate acrylamide monomers inducing them to react with other acrylamide monomers forming long chains.  Used to separate most proteins and small oligonucleotides because of the presence of small pores.
  7. 7. MATERIAL REQUIRED FOR GEL ELECTROPHORESIS         Electrophoresis chamber gel Gel casting tray Buffer Staining agent (dye) A comb DNA ladder Sample to be separate
  8. 8. GEL ELECTROPHORESIS EQUIPMENT
  9. 9. GEL COSTING TRAYS  available in a variety of sizes and composed of UV-transparent plastic.  The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
  10. 10. APPLIED VOLTAGE  voltage, rate of migration The higher the voltage, the more quickly the gel runs But if voltage is too high, gel melts The best separation will apply voltage at no more than 5V/cm of gel length.
  11. 11. BUFFERS During electrophoresis water undergoes hydrolysis : H 2 O  H + OHBuffers prevent the pH from changing by reacting with the H+ or OH- products Most common buffer used is called TRIS [tris(hydroxymethyl)aminomethane]
  12. 12. BUFFERS Another compound is added to make Tris an effective buffer — either boric or acetic acid Another compound is added to bind metals EDTA The buffer is either TBE or TAE  TBE is made with Tris/Boric Acid/EDTA  TAE is made with Tris/Acetic Acid/ EDTA
  13. 13. ETHIDIUM BROMIDE  The standard concentration used in staining DNA in gels is 0.5-1ug/mL  Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels.  Inserting itself between the base pairs in the double helix
  14. 14. STAINING OF DNA  UV absorbance maxima at 300 and 360 nm and emission maxima at 590 nm.  Detection limit of bound DNA is 0.5 -5 ng/band.  ethidium bromide is mutagenic so care must be taken while handling the dye.  Other alternatives for ethidium bromide :  Methylene blue  Syber safe  xylene cyanol  bromphenol blue
  15. 15. A COMB  A comb is placed in the liquid agarose after it has been poured  Removing the comb from the hardened gel produces a series of wells used to load the DNA
  16. 16. METHOD FOR ELECTROPHORESIS Prepare agarose gel Melt, cool and add Ethidium Bromide. Mix thoroughly Pour into casting tray with comb and allow to solidify Add running buffer, load samples and marker Run gel at constant voltage until band separation occurs View DNA on UV light box and show results
  17. 17. APPLICATION Electrophoresis is employed in biochemical and clinical field. In the study of protein mixtures Antigen antibody reactions In fractioning protein. In analysis of lipoprotein Hemoglobin
  18. 18. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids, insulin. In food industry In combination with autoradiography
  19. 19. THANK YOU
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