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).