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        RAPD PCR Colony Miniprep

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        993
         
         
        1. Edit the program or pick up a program (No 10) if the program has been set.
          Reaction time for RAPD PCR .
          1 cycle : Denature 95 C for 2 min
          45 cycle of
          Denature 94 C 15 sec
          Anneal 40 C 30 sec
          Extend 72 C 90 sec
          1 cycle of 72 C for 5 min
          4 C hold
        2. Inoculate a touched-bacterial colony with a sterile toothpick into 100 µl of sterile H2O and voltex the tube.
        3. Boil (95 C) for 5 min in the water bath
        4. Cool on the ice for 1-2 min and voltex.
        5. Spin (14 K, 5 min).
        6. Withdraw 2 µl of the sample (supernatent), add to 16 µl H2O in the 0.2 ml tube, and add a mixture of 2.5 µl 10X PCR buffer, 2 µl 2.5 µM primer , 2 µl 2.5 mM dNTP (2.5 mM each of dATP, dCTP, dGTP, and dTTP) and 0.5 U AmpliTaq.
        7. Seal the tube cap.
        8. RUN to start the program.
        9. After the whole cycle, add 2.5 µl stopping dye.
        10. Withdraw 10 or 12.5 µl of sample with a P-20 pipet and subject to electrophoresis (1.2 or 1.4 % agarose gel, 160 V) for 1 hr.
        Reference: Zon, L. I., Dorfman, D. M., Orkin, S. H. 1989. The Polymerase Chain Reaction colony miniprep. Biotechnique 77: 696-698. <center> <p>  </p> </center>
        上一篇:RFLP和RAPD技术原理和操作步骤   下一篇:RAPD操作要点
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