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        Colony PCR 菌落PCR【Upstate Medical University】

        互联网

        1995
        Amberg Lab ,Upstate Medical University http://www.upstate.edu/biochem/amberg/protocols/colony_pcr.html

        This procedure will work for both yeast and E. coli:

        Take a small colony and suspend it in 5ul of H2O in a PCR tube. Heat for 5 min at 95¡C and then spin the condensation down in a microfuge. Set up the PCR reaction as follows:

         

        • 5ul H2O cells
        • 5ul 5uM primer2
        • 5ul 10XTaq Buffer
        • 5ul 2mM dNTPs
        • 0.5ul 10 mg/ml acetylated BSA
        • 1ul Taq DNA polymerase
        • 23.5 ul H2O

        PCR Conditions: 94¡C x 4min. then 35 cycles of: (94¡C x 1 min then 55¡C x 1min then 72¡C x 3min) followed by 72¡C x 20 min and a 4¡C soak. (5ul run out on a mini gel should be sufficient to see product.)

         

        • 10X TAQ Buffer:
        • 0.2M TRIS pH8.3
        • 15mM MgCl2
        • 0.25M KCl
        • 0.5% TWEEN 20

        <center> <p>  </p> </center>
        上一篇:Disruption by Fusion PCR【Upstate Medical University】   下一篇:TAIL PCR Protocol
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