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Gel electrophorosis final

Gel electrophorosis final






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    Gel electrophorosis final Gel electrophorosis final Presentation Transcript

    • Gel Electrophoresis NAYANA.P and Jitendra Kumar Dept OF FRM COLLEGE OF FISHERIES jitenderanduat@gmail.com
    • Introduction Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. jitenderanduat@gmail.com
    • Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving jitenderanduat@gmail.com
    • Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP,  PCR, cloning, DNA sequencing, or  Southern blotting for further characterization. jitenderanduat@gmail.com
    • Types of gel Agarose Polyacrylamide Starch jitenderanduat@gmail.com
    • Agarose  gels are easily cast and handled compared to other matrices, because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator. advantages: it is used for the separation of DNA fragments ranging from 50 base pair to several millions of bases using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel jitenderanduat@gmail.com
    • The disadvantage of higher concentrations is the long run times (sometimes days). Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger jitenderanduat@gmail.com
    • Polyacrylamide Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins Pore size is controlled by controlling the concentrations of acrylamide and bisacrylamide powder used in creating a gel. jitenderanduat@gmail.com
    • Starch Partially hydrolyzed potato starch makes for another non-toxic medium for protein electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualized using Napthal Black or Amido Black staining. jitenderanduat@gmail.com
    • How does it work? • DNA is cut into smaller fragments. • Loading dye is used to indicate the fragments of DNA are behind the dye • The negative DNA molecule is attracted to the positive electrode. • The smallest fragments move the greatest distance. jitenderanduat@gmail.com
    • Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field ◦ DNA is negatively charged ◦ when it’s in an electrical field it moves toward the positive side DNA – →→→→→→→ “swimming through Jello” + jitenderanduat@gmail.com
    • Gel electrophoresis DNA moves in an electrical field… ◦ so how does that help you compare DNA fragments?  size of DNA fragment affects how far it travels  small pieces travel farther  large pieces travel slower & lag behind DNA – →→→→→→→ “swimming through Jello” + jitenderanduat@gmail.com
    • Gel Electrophoresis DNA & restriction enzyme longer fragments wells power source gel shorter fragments + completed gel jitenderanduat@gmail.com
    • fragments of DNA separate out based on size Running a gel cut DNA with restriction enzymes 1 2 3 Stain DNA ◦ ethidium bromide binds to DNA ◦ fluoresces under UV light jitenderanduat@gmail.com
    • Procedure Remove comb and observe wells. Place carbon paper in each end of the tray. Cover with buffer, making sure the allow buffer to overflow into each end of the tray. Load gels. Connect the electrodes. Turn on power supply. Allow gels to run – make sure you see bubbles coming from the electrodes. jitenderanduat@gmail.com
    • PROCEDURE (CONTINUED) It will take about 30 minutes for the gel to run. Turn off power supply and remove electrodes. Pour off buffer into the designated container. Carefully remove gel from gel box and place in glad container and cover with stain. Store in appropriate location. jitenderanduat@gmail.com
    • Applications Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in  restriction mapping of cloned DNA. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of restricted genomic DNA Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemist ry. jitenderanduat@gmail.com
    • Proteins can also be run on gels. Most commonly proteins are run on gels made of polyacrylamide in the presence of SDS Scientific dyes can also be separated by gel electrophoresis. jitenderanduat@gmail.com
    • jitenderanduat@gmail.com