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        SDS-polyacrylamide gel electrophoresis

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        1640

         

         

        SOLUTIONS

        30% acrylamide mix 29% (w/v) acrylamide
        1% (w/v) N,N''- methylenebisacrylamide
        5x Electrophoresis buffer Tris base: 15.1 g
        Glycice: 94 g
        SDS: 5 g
        H2 O upto1 liter
        4x Resolving-gel buffer 1.5M Tris-Cl (pH 8.8)
        8x Stacking-gel buffer 1M Tris-Cl (pH 6.8)
        1x SDS gel-loading buffer 50mM Tris-Cl (pH 6.8)
        100mM dithiothreitol
        2% SDS
        0.1% bromophenol blue
        10% glycerol



        RESOLVING GEL MIXTURE
        Resolving Gel (30ml) 6% 8% 10% 12% 15%
        H2 O 16.2 14.2 12.2 10.2 7.2
        30% acrylamide mix 6.0 8.0 10 12 15
        4x resolving-gel buffer 7.5 7.5 7.5 7.5 7.5
        20% SDS 0.15 0.15 0.15 0.15 0.15
        20% APS 0.15 0.15 0.15 0.15 0.15
        TEMED 0.024 0.018 0.012 0.012 0.012

        Resolving Gel (10ml) 6% 8% 10% 12% 15%
        H2 O 5.4 4.7 4.1 3.4 2.4
        30% acrylamide mix 2.0 2.7 3.3 4 5
        4x resolving-gel buffer 2.5 2.5 2.5 2.5 2.5
        20% SDS 0.05 0.05 0.05 0.05 0.05
        20% APS 0.05 0.05 0.05 0.05 0.05
        TEMED 0.008 0.006 0.004 0.004 0.004



        STACKING GEL MIXTURE
        Stacking Gel (10ml)    
        H2 O 6.9 ml
        30% acrylamide mix 1.7 ml
        8x stacking-gel buffer 1.25 ml
        20% SDS 0.05 ml
        20% APS 0.05 ml
        TEMED 0.01 ml



        SOLUTIONS FOR STAINING AND DESTAINING
        Staining solution methanol 90 ml
        H2 O 90 ml
        acetic acid 20 ml
        Coomassie Brilliant Blue R250 0.25 g
        Dissolve well and filtrate through a filter paper.
        Destaining solution isopropanol 12.5%
        glacial acetic acid 10%
        Addition of small pieces of used X-ray film with the
        gel will accelerate the destaining process.



        SOLUTION FOR DRYING

         

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