Sanjay patilM- PHARMA [QA]

SOPS RGPV

GEL ELECTROPHORSIS

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

ELETOPHORSIS

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.

GEL ELECTROPHORESIS

Charged molecules are separated based on their electrical charge and size.

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.

TYPE OF 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.

POLYACRYLAMIDE GEL

Electrophoresis chamber gel Gel casting tray Buffer Staining agent (dye) A comb DNA ladder Sample to be separate

MATERIAL REQUIRED FOR GEL ELECTROPHORESIS

GEL ELECTROPHORESIS EQUIPMENT

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.

GEL COSTING TRAYS

voltage, rate of migrationThe higher the voltage, the more

quickly the gel runsBut if voltage is too high, gel meltsThe best separation will apply voltage

at no more than 5V/cm of gel length.

APPLIED VOLTAGE

During electrophoresis water undergoes hydrolysis : H2O 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]

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

BUFFERS

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

ETHIDIUM BROMIDE

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

STAINING OF DNA

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

A COMB

Prepare agarose gelMelt, 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

METHOD FOR ELECTROPHORESIS

Electrophoresis is employed in biochemical and clinical field.

In the study of protein mixturesAntigen antibody reactionsIn fractioning protein.In analysis of lipoproteinHemoglobin

APPLICATION

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

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