Agarose Gel Electrophoresis Lab - PDF

Genetics and Molecular Biology 3rd stage MLT Department Agarose Gel Electrophoresis  Why electrophoresis? To separate DNA fragments from each other To determine the sizes of DNAfragments To determine the presence or amount of DNA Elec...

Genetics and Molecular Biology 3rd stage MLT Department Agarose Gel Electrophoresis  Why electrophoresis? To separate DNA fragments from each other To determine the sizes of DNAfragments To determine the presence or amount of DNA Electrophoresis separates To analyze restriction negatively charged nucleic acid molecules by applying an electric digestion products field, causing them to move through an agarose matrix based  Introduction Of Agarose Gel Electrophoresis Agarose gel electrophorresis is a method to separate DNAor RNAmolecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field(electrophoresis). Shorter molecules move faster and migrate faster than longer ones.  Principle of electrophoresis powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes convenient analytical method for determining the size of DNAmolecules in the range of 500 to 30,000 base pairs. employs electromotive force to movemolecules through a porous gel  Principle (cont.) separates molecules from each other on the basis of – size and/or – charge and/or – shape basis of separation depends on how the sample and gel are prepared  Positive Molecules Analyze Size Charge Separation Identify Separation Mixture of Charged Purify Molecules Negative Molecules Material required for agarosegel electrophoresis - Electrophoresis chamber - Agarose gel - Gel casting tray - Buffer - Staining agent (dye) - Comb - DNAladder - Sample to be separate Electrophoresis Equipment  What is Agarose ? A linear carbohydrate polymer extracted from seaweed , agarobiose forms a porous matrix as it gels – shifts from random coil in solution to structure in which chains are bundled into doublehelices  TYPESOFAGAROSE Standard Agarose - LE -Gels at 35-38oC; Melts at 90-95oC - Becomes opaque at high concentrations Low Melting Agarose (NuSieve) -Gels at 35oC; Melts at 65oC - Often used to isolate DNAfragments from gel - Intermediate forms or combinations ofLE and NuSieve can provide sturdy, translucent gels at high agarose concentrations. Concentrations of agaroseused %Agarose (w/v) Size Range (kb pairs)for Optimal Separation 0.5 2-30 0.75 0.7-20 1.0 0.5-10 1.5 0.2-3 2.0 0.1-2 Gel Casting 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.  Applied voltage  voltage,  rate of migration The higher the voltage, the more quicklythe gel runs But if voltage is toohigh, gel melts The best separation will apply voltage atno more than 5V/cm of gellength.  Buffers During electrophoresis water undergoes hydrolysis : H2O H + OH- Buffers prevent the pH from changingby reacting with the H+ or OH- products Most common buffer used is called TRIS – [tris - (hydroxymethyl)aminomethane]  Buffers (cont.) Another compound is added to make Trisan effective buffer —either boric or aceticacid Another compound is added to bindmetals EDTA The buffer is either TBEorTAE TBEis made with Tris/BoricAcid/EDTA TAEis made with Tris/Acetic Acid/EDTA  Staining of DNA To make DNAfragments visible after electrophoresis, the DNAmust be stained The favorite—ethidium bromide When bound to DNAit fluoresces under ultraviolet light (reddish –orangecolour) Convenient because it can be addeddirectly to the gel Sensitive—detects 0.1ug of DNA 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 basesof nucleic acids and allows very convenient detection of DNA fragments in gels. Inserting itself between the base pairs in the double helix  Staining of DNA(cont.) UV absorbance maxima at 300 and 360 nm and emission maxima at 590 nm. Detection limit of bound DNA is 0.5-5ng/band. ethidium bromide is mutagenic so care must be taken while handling the dye. Othe alternatives for ethidium bromide: Methylene blue Syber safe xylene cyanol bromphenol blue  A Comb A comb is placed in the liquid agarose after it has been poured Removing the comb from the hardenedgel produces a series of wells used to load the DNA DNA ladder provide a standard for sizing DNA fragments It is a solution of DNA molecules of different length DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spacedsized samples which when run onan agarose gel looks like a "ladder". Sample preparation  DNA is negatively charged. When placed in an electrical field, DNA will migrate towardthe positive pole (anode). An agarose gel is used to slow the movement of DNA andseparate by size. H O 2 DNA - + Power How fast will the DNAmigrate? strength of the electrical field, buffer, density of agarosegel… Size of the DNA! *Small DNAmove faster than large DNA …gel electrophoresis separates DNAaccording to size DNA small large - + Power  Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight. Thank You..