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Team:TU Delft/protocols/ligation
From 2010.igem.org
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| - | + | =Ligation= | |
| - | + | ''Materials:'' | |
| + | - digested plasmid DNA or PCR product | ||
| + | |||
| + | - T4 ligation buffer (10x) (Fermentas or BioLabs) | ||
| + | |||
| + | - T4 ligase (Fermentas or BioLabs) | ||
| + | |||
| + | - H2O | ||
| + | |||
| + | - water bath at 16 °C | ||
| + | |||
| + | |||
| + | ''Protocol:'' | ||
| + | |||
| + | Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. | ||
Reaction for one sample: | Reaction for one sample: | ||
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|- | |- | ||
|T4 Ligation buffer (10×) | |T4 Ligation buffer (10×) | ||
| - | | | + | |x μL (for 1×) |
|- | |- | ||
|T4 Ligase | |T4 Ligase | ||
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|- | |- | ||
| | | | ||
| - | | | + | |10-15 μL |
|} | |} | ||
| + | The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. | ||
| + | For transformation use circa half of the ligation mix. | ||
Transform circa half of the ligation mix. Incubate at 16 °C o/n | Transform circa half of the ligation mix. Incubate at 16 °C o/n | ||
Latest revision as of 18:52, 12 September 2010
Ligation
Materials:
- digested plasmid DNA or PCR product
- T4 ligation buffer (10x) (Fermentas or BioLabs)
- T4 ligase (Fermentas or BioLabs)
- H2O
- water bath at 16 °C
Protocol:
Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.
Reaction for one sample:
| DNA insert | x μL |
| DNA vector | x μL |
| T4 Ligation buffer (10×) | x μL (for 1×) |
| T4 Ligase | 1.0 μL |
| H2O | x μL |
| 10-15 μL |
The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix.
Transform circa half of the ligation mix. Incubate at 16 °C o/n
