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        Polymerase Chain Reaction--热启动PCR

        互联网

        1459

        This procedure is a "hot start" PCR cycle for use with large primers. PCR mass-produces DNA contained in the source plasmid between the two primer-binding sequences, eventually producing linear DNA starting with Primer 1, continuing with the source sequence, and ending with the complementary sequence to Primer 2.

        --------------------------------------------------------------------------------

        Materials

        10 mM dNTP mix [Boehringer Mannheim, cat # 1581 295]
        10X PCR Buffer w/ 15 mM MgCl2 [Boehringer Mannheim, cat # 1759 167]
        PCR Enzyme: Expand HF PCR System [Boehringer Mannheim, cat # 1732 641]
        150 µM Primer 1
        150 µM Primer 2
        Source plasmid
        Mineral oil
        Chloroform: ACS Reagent Grade
        PCR Thermocycler
        Procedure

        Prepare Master Mix 1 in a 500 µL tube on ice:
        2 µL dNTP solution
        2 µL dilute Primer 1 (dilute 1:10 in sterile dH2O)
        2 µL dilute Primer 2 (dilute 1:10 in sterile dH2O)
        4 µL source plasmid (adjust amount as necessary)
        40 µL sterile dH2O
        Prepare Master Mix 2 in a 500 µL tube on ice:
        10 µL 10X PCR Buffer
        0.75 µL PCR Enzyme
        39 µL sterile dH2O
        Immediately before starting the PCR cycle, add MM2 to MM1; to prevent evaporation, place two drops of mineral oil on top of the solution
        Run the following PCR cycle:
        First cycle: 5 minutes at 95 °C
        Next thirty cycles: 1 minute at 94 °C, 2 minutes at 55°C, 3 minutes at 72 °C
        Hold at 4 °C
        Add 150 µL of Chloroform to the PCR product to remove mineral oil; gently invert to mix
        Transfer the aqueous "bubble" (floating on top) to a fresh 500 µL tube with a gel-loading tip on a 20 µL micropipet
        Store PCR Product at -20 °C

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