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  • Cloning with USER Enzyme

    Protocol

    1. Amplify your target DNA using Taq DNA Polymerase and uracil-containing primers.
    2. Assembly Reaction:
      10 μl crude PCR sample
       1 μl Linearized pNEB206A (20ng)
       1 μl USER Enzyme (1 unit)
      -----------------------------------------
      12 μl total volume
    3. Incubate for 15 minutes at 37°C.
    4. Incubate for 15 minutes at room temperature.
    5. Transform chemically competent E. coli cells with 2-12 μl of the assembly reaction from Step 4.

      The cloned insert can be removed by BbvCI cleavage (NEB #R0601).