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        使用 Platinum® Taq DNA 聚合酶 (Platinum® Taq DNA Polymerase),对靶序列进行定性验证

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        实验步骤

         

        The procedure on the following page is suggested as a guideline and starting point when using Platinum® Taq  DNA Polymerase in any PCR amplification.  Optimal reaction conditions (incubation times and temperatures, concentration of Platinum® Taq  DNA Polymerase, primers, MgCl2, and template DNA) vary and need to be optimized.  Reaction size may be altered to suit user preferences.                                                                                              

        1.        Add the following components to a sterile 0.5-ml microcentrifuge tube:
                 10X PCR Buffer, Minus Mg       5  µl

        10 mM dNTP mixture              1  µl    

        50 mM MgCl2                1.5 µl

        Primer mix (10 µM each)        1 µl

        Template DNA                ≥ 1 µl  (as required)

        Platinum® Taq DNA Polymerase  0.2 µl

        Autoclaved, distilled water       to 50 µl

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

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

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

        5.         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

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

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

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