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        High Specificity PCR Amplification Using AccuPrime Taq DNA Polymerase

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        实验步骤

         

        1.        PCR Precautions

        1)        Add the following components to a sterile thin walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice:

        For Small Genomic DNA ( < 200 bp), Plasmids, or cDNA):

                                                                          Table 1

        Component

        10-µl Reaction

        25-µl Reaction

        50-µl Reaction

        10X AccuPrime™  PCR Buffer I

        1 µl

        2.5 µl

        5 µl

        Primer Mix (10 µM each)

        0.2 µl

        0.5 µl

        1 µl

        Template DNA

        10 pg–200 ng

        10 pg–200 ng

        10 pg–200 ng

        AccuPrime™  Taq  DNA Polymerase

        0.25 µl

        0.5 µl

        1 µl

        Autoclaved distilled water

        To 10 µl

        To 25 µl

        To 50 µl

        For Genomic DNA (200 bp-4 kb):

                                                                         Table 2

        Component

        10-µl Reaction

        25-µl Reaction

        50-µl Reaction

        10X AccuPrime™  PCR Buffer II

        1 µl

        2.5 µl

        5 µl

        Primer Mix (10 µM each)

        0.2 µl

        0.5 µl

        1 µl

        Template DNA

        1–200 ng

        1–200 ng

        1–200 ng

        AccuPrime™  Taq  DNA Polymerase

        0.25 µl

        0.5 µl

        1 µl

        Autoclaved distilled water

        To 10 µl

        To 25 µl

        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 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 15-30 s

              Anneal:        55°C-60°C for 15-30 s

              Extend:        68°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 amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.

        2.        Multiplex PCR Protocol

        1)        Add the following components to a sterile, thin-walled 0.25-ml or 0.5-ml PCR tube either at room temperature or on ice:

                                                                    Table 3

        Component

        Amount

        10X AccuPrime™  PCR Buffer I (for genomic DNA <200 bp, cDNA, or plasmids) or

        10X AccuPrime™  PCR Buffer II (for genomic DNA 200 bp–4 kb)

        5 µl

        Primer mix (10 µM each)

        1 µl each (0.2 µM each)

        Template DNA

        100-200 ng

        AccuPrime™   Taq   DNA Polymerasundefined

        1-2.5 µl

        Autoclaved, distilled water

        to 50 µl

         

        2)        Continue with steps 2-7 of the General Protocol.

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