"

From 2012.igem.org

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

1. Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.

1. 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.