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Team:WashU/Protocols/Ligation
From 2012.igem.org
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=General Ligation Protocol= | =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 | + | (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. | |
Revision as of 20:51, 13 June 2012
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.
