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        Polymerase Chain Reaction (PCR)【University of Texas at Austin】

        互联网

        1254
        Kitto Lab, The University of Texas at Austin PCR .html">http://research.cm.utexas.edu/bkitto/Kittolabpage/Protocols/Microbiology/PCR .html

        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

        1. 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
        2. 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
        3. Immediately before starting the PCR cycle, add MM2 to MM1; to prevent evaporation, place two drops of mineral oil on top of the solution
        4. 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
        5. Add 150 µL of Chloroform to the PCR product to remove mineral oil; gently invert to mix
        6. Transfer the aqueous "bubble" (floating on top) to a fresh 500 µL tube with a gel-loading tip on a 20 µL micropipet
        7. Store PCR Product at -20 °C

        <center> <p>  </p> </center>
        上一篇:Long PCR Reagents and Guidelines长距离PCR反应溶液和反应条件   下一篇:Disruption by Fusion PCR【Upstate Medical University】
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