General Ligation Protocol

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.