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        RNA电泳实验方法

        互联网

        1664

         

        Polyacrylamide Gel Electrophoresis (PAGE)

        for use withRibonuclease Protection Assay (RPA):

        1. Making the Gel:

          5% Denaturing gel for Ribonuclease Protection Assay:

                 Urea,high quality                7.2 g

                     10X TBE                      1.5 ml

                 30% acrylamide/bis               2.5 ml

           or use ready to go 40% acrylamide      1.9 ml

                    Deionized H2O              up to 15 ml

        Stir at room temperature until urea has dissolved then add:

                      10% APS                      120 ul

                       TEMED                        16 ul

        For an 8% gel use 4 ml of 30 % acrylamide (3 ml of 40%)

        For a 10% gel use 5 ml of 30% acrylamide (3.75 ml of 40%)

        2. For denaturing gels only, heat all tubes for 3-4 minutes at 90’C.

        3. Vortex, spin down and put on ice.

        4. Pre run gel for 5 minutes, rinse out well with buffer then immediately load gel.

        5.Run for approximately 1 hour at 250 volts in TBE buffer.

        6. Transfer to positively charged nylon membrane according to manufactures protocol.

        Recommended % acrylamide: Size of Bands: Bromophenol Blue:

        (Denaturing gels)

        4 >250 30

        6 60-250 25

        7 40-120 20

        10 20-60 10

         

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