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        Addition of 3-A Overhangs (A-Tailing) to PCR Product

        互联网

        1183

         

      • After amplification with a proofreading polymerase, place samples on ice and add 0.7-1 unit of Taq polymerase per tube directly into the PCR reaction tube. Mix well.
      • Incubate at 72°C for 8-10 minutes.
      • Place the reaction on ice and use it immediately for ligation reaction with a TA cloning vector such as the TOPO TA cloning vector from Invitrogen.
      • Alternatively, the PCR product can be purified before used in ligation reaction as follows.
      • Extract reaction immediately with an equal volume of phenol-chloroform to remove all of the polymerases.
      • Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate and 2X volume of 100% ethanol.
      • Centrifuge at maximum speed (14,000 rpm) for 5 minutes at room temperature to pellet the DNA.
      • Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry.
      • Resuspend the pellet in TE buffer to the starting volume of the PCR amplification reaction. The PCR amplification product is now ready
        for ligation into a TA vector.

        •  

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