1.Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. (Upstream Part digestion: 2 µl), (Downstream Part digestion:2 µl), (Destination Plasmid digestion:2 µl), (10X T4 DNA Ligase Buffer:2 µl), (T4 DNA Ligase: 1 µl), (H2O:11 µl)
2.Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.
3.Transform 2 μl of the ligation product into 50 μl of competent E. coli cells (or other suitable host strain). Select using the antibiotic corresponding to the Destination Plasmid.
Thanks to NEB for the above instructions. The original source can be found here: [http://www.neb.com/nebecomm/products_intl/protocol658.asp]
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