Cloning with Thermolabile USER Enzyme Protocol (M5507)

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:
Crude PCR Sample
 10 µl
Linearized pNEB206A (20 ng)
 1 µl
Thermolabile USER Enzyme (1 unit)
 1 µl
 12 µl

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).