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        Platinum® Taq Antibody

        互联网

        2489

        实验步骤

         

        1.        Measure out the amount of Taq DNA polymerase needed. When using a reaction cocktail, determine the total number of units in the mix.

        2.        Add an amount of “equivalent units” of Platinum® Taq Antibody to an equal amount of Taq DNA polymerase units. ForTaq DNA polymerase at 5 unit per μl, prepare a 1:1 mixture. Mix well using a Vortex Mixer.

        3.        Add the following components to a sterile 0.5-ml microcentrifuge tube:

                                                                Table 1

        Components

        Volume

        Final Concentration

        10X PCR Buffer, Minus Mg

        5 μl

        1X

        10 mM dNTP mixture

        1 μl

        0.2 mM each

        50 mM MgCl2

        1.5 μl

        1.5 mM

        Primer mix (10 μM each)

        1 μl

        0.2 μM each

        Template DNA

        ≥1 μl

        (as required)

        Platinum® Taq  Antibody: 
        Taq  DNA polymerase

        1 μl

        2.5 units (or as required)

        Autoclaved, distilled water

        to 50 μl

        Not applicable

         

        4.        Mix contents of the tubes and overlay with 50 μl of mineral or silicone oil, if necessary.

        5.        Cap the tubes and centrifuge briefly to collect the contents

        6.        Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min to completely denature the template and activate the enzyme.

        7.        Perform 25-35 cycles of PCR amplification as follows: Denature 94°C for 30 s Anneal 55°C for 30 s Extend 72°C for 1 min per kb

        8.        Maintain the reaction at 4°C after cycling. The samples can be stored at -20°C until use.

        9.        Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards

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