Gibson Assembly Cloning

Gibson assembly requires a vector backbone and one or more inserts that have been PCR amplified. The inserts should have 15-20 base pairs of overlap at ligation sites, so primers used to amplify the inserts should contain a tail overhang with this homology. The vector backbone should be linear. A Gibson reaction uses a master mix that can be homemade or commercially purchased. Add the appropriate amount of master mix, then your vector and insert at 2-3 fold molar excess for a 20 µl reaction. Exact ratios will need to be optimized based on the size of your vector and inserts and how many inserts you have. Incubate this reaction at 50°C for 60 minutes. Then transform this reaction into DH5 alpha (or competent cell of choice) and plate on LB agar with appropriate antibiotic.