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Gel Electrophoresis

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    1. Presented by: Subha Ramnarayan Date: 03/07/2011 Gel Electrophoresis

    3. Theory/Principle Electrophoretic technique has been developed based on the following factor: Electrophoretic mobility When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.

    4. Theory/Principle Sample is placed into the gel matrix medium Subjected to an electric field. Molecules move at different rates-smaller molecules move faster than larger molecules After the specified amount of time has elapsed, the current is switched off Mixture is stained with a dye that binds to proteins or nucleic acids and readings are taken.

    5.

    6. Theory/Principle (continued) Example of SDS-PAGE of proteins visualized by autoradiography.

    7. Instrumentation

    8. Types of Gel Electrophoresis SDS-PAGE: (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis): SDS denatures proteins prior to gel electrophoresis. Agarose Gel electrophoresis: Agarose forms a complex matrix through which nucleotides can migrate. Sequencing Gel:. Allows isolation of DNA sequences that differ by as little as one base pair. Electrofocusing gel: For the separation of protein molecules without denaturing them; proteins migrate based upon their native charge.

    9. Applications Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil Goal: To monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. Technique: DGGE was done on samples from polluted soil and on unpolluted soil.

    11. Applications (continued) Conclusion: DGGE method serves as a diagnostic tool to quickly monitor and compare overall ciliate community structures in polluted and unpolluted soils.

    12. Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes Goal: To analyze the integrity of nucleoprotein complexes by checking the change in the values of Re (effective radius) and µ'0 (electrophoretic mobility) values. Technique: Single and multiple loading methods. Bacteriophage T3 is used as an internal control to calculate the pore size of the agarose gels in each lane. µ/µ'0=(1-Re/Pe)2 Results: No significant change in Re and µ'0 values due to T3 bands: Applications (continued)

    16. Conclusion/Advantages/Disadvantages Overall Conclusion: Gel electrophoresis is used in separating DNA, RNA and protein molecules. Has analytical and preparative applications. Advantages: Agarose Gel electrophoresis: Gel is easily poured, does not denature the samples. The samples can be recovered. Polyacrylamide gel electrophoresis: For fragments below the size range of agarose gel electrophoresis. In PAGE up to 10 micrograms of DNA can be loaded into a single well (1 cm x 1 mm) without significant loss of resolution. Polyacrylamide contains few inhibitors of enzymatic reactions. Is an ideal gel system from which to isolate DNA fragments for subcloning and other molecular biological techniques

    17. Disadvantages: Agarose Gel electrophoresis: Gels can melt during electrophoresis The buffer can become exhausted Different forms of genetic material may run in unpredictable forms Polyacrylamide gel electrophoresis: Mobility of the fragments can be affected by base composition -making accurate sizing of bands a problem. Polyacrylamide quenches fluorescence, making bands containing less than 25 ng difficult to visualize with ethidium bromide staining. Conclusion/Advantages/Disadvantages(Contd)

    18. Bibliography http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/principles.html http://en.wikipedia.org/wiki/Gel_electrophoresis http://en.wikipedia.org/wiki/File:SDSPAGE.png http://web.utk.edu/~khughes/GEL/sld013.htm http://learn.genetics.utah.edu/content/labs/gel/ http://www.life.illinois.edu/molbio/geldigest/electro.html http://www.molecularstation.com/molecular-biology-techniques/gel-electrophoresis/ http://biotech.matcmadison.edu/resources/proteins/labManual/chapter_5/procedure5_3.htm http://www.ehow.com/list_7194999_types-gel-electrophoresis.html Adkins, Nicholas L, J Adam Hall, and Philippe T Georgel. "The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes." Journal of Biochemical and Biophysical Methods 70.5 (2007): 721-726 . ScienceDirect. Web. 6 Mar. 2011. <http://sciencedirect.com>.

    19. Jousset, Alexandre, et al. "Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil ." Science of The Total Environment 408.5 (2010): 1221-1225 . ScienceDirect. Web. 6 Mar. 2011. <http://sciencedirect.com>. Bibliography(Contd)