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Team:WashU/Protocols/Ligation
From 2012.igem.org
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=General Ligation Protocol= | =General Ligation Protocol= | ||
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#Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. | #Ligate the Upstream and Downstream Parts into the digested Destination Plasmid. | ||
| - | Upstream Part digestion: 2 µl | + | Upstream Part digestion: 2 µl |
| - | Downstream Part digestion: 2 µl | + | Downstream Part digestion: 2 µl |
| - | Destination Plasmid digestion: 2 µl | + | Destination Plasmid digestion: 2 µl |
| - | 10X T4 DNA Ligase Buffer: 2 µl | + | 10X T4 DNA Ligase Buffer: 2 µl |
| - | T4 DNA Ligase: | + | T4 DNA Ligase: 1 µl |
| - | H2O: | + | H2O: 11 µl |
#Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes. | #Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes. | ||
#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. | #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 20:46, 13 June 2012
General Ligation Protocol
- 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
- Incubate at room temperature for 10 minutes and then heat inactivate at 80°C for 20 minutes.
- 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.
