Cloning with USER® Enzyme

Protocol

  1. Amplify your target DNA using Taq DNA Polymerase, or any other DNA Polymerase which is not inhibited by a dU-containing template, 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).