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Sds page and agarose presentation.
 

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    Sds page and agarose presentation. Sds page and agarose presentation. Presentation Transcript

    • Agarose and Polyacrylamine Gel Electrophoresis Khrystall K. Ramos CallejasDepartment of Biology Gretel S. Montañez PróspereUniversity of Puerto Rico at Cayey Luis Pérez SotoRISE Program Wilmarie Morales SotoMarch 16, 2012
    • WHAT IS GEL ELECTROPHORESISElectrophoresis is the term used for the procedure where under the influence of voltage, a charged particle moves.It is a standard method for separation, identification, analysis and purification of: DNA molecules protein molecules
    • Electrophoresis consists of the migration of a charged molecules under the influence of electric field (from negative to positive).A buffer solution is use to conduct electricity through the whole setup of the gel electrophoresis.The molecule will migrate through the gel depending upon the size and shape.
    • Gel electrophoresis is used: Forensics Molecular biology Genetics Microbiology BiochemistryThe results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device; analyzing the intensity of the band or the measure of the spot of interest.
    • TYPES OF GELS:1. Agarose*2. Polyacrylamide*3. Starch
    • AGAROSE GEL ELECTROPHORESISAgarose is a linear polymer extracted from seaweed that forms a gel matrix by hydrogen- bonding when heated in a buffer and allowed to cool.The agarose gel is used to separate DNA and RNA fragments.Agarose gels separate DNA fragments differing by a hundred or more base pairs.
    • DNA has negative charge so it migrates towards the positive end.This is due to its double helical physical structure, which contains a phosphate backbone.
    • The density and porosity of the gel matrix is determined by the concentration of agarose used.The grater the agarose concentration, the smaller the pores created in the gel matrix, the more difficult it is for larger DNA molecules to move through. Agarose % Optimum Resolution for DNA 0.5 1,000-30,000bp 0.7 800-12,000bp 1.0 500-10,000bp 1.2 400-7,000bp 1.5 200-3,000bp 2.0 50-2,000bp
    • POLYACRYLAMIDE GEL ELECTROPHORESISLike Agarose Gels, Polyacrylamide gels are used to separate protein molecules by shape, size and charge.Polyacrylamide is a polymer of acrylamide monomers.
    • Polyacrylamide is specifically used for proteins because it provides the protein with an environment where it will not become denatured.Allowing different sized proteins to move at different rates.
    • Since we are trying to separate many different protein molecules of a variety of shapes and sizes, we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure.
    • To have proteins with linear structures we use sodium dodecyl sulfate (SDS).SDS is a detergent that can dissolve hydrophobic molecules, resulting in proteins with linear structures.
    • Another problem we face with proteins is that they do not have a specific charge.This is another reason why SDS is important. SDS has a negative charge and by dissolving the protein in it, the protein becomes negatively charged.Allowing it to run properly through the gel (from negative to positive).
    • Get your sample obtained from previous purifying technique (i.e. PCR) Load BufferSet up gel, removecomb Load Sample Run GelStain and look at withUV light
    • APPLICATIONS OF GELS:Estimation of the size of DNA and protein molecules.Analysis of PCR products, i.e. in molecular genetic diagnosis or genetic fingerprintingSeparation of restricted genomic DNA or of RNA.