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Team:TU Delft/protocols/ligation
From 2010.igem.org
(Difference between revisions)
| Line 5: | Line 5: | ||
- digested plasmid DNA or PCR product | - digested plasmid DNA or PCR product | ||
| - | - T4 ligation buffer (10x) (Fermentas) | + | - T4 ligation buffer (10x) (Fermentas or BioLabs) |
| - | - T4 ligase (Fermentas) | + | - T4 ligase (Fermentas or BioLabs) |
- H2O | - H2O | ||
| Line 27: | Line 27: | ||
|- | |- | ||
|T4 Ligation buffer (10×) | |T4 Ligation buffer (10×) | ||
| - | | | + | |x μL (for 1×) |
|- | |- | ||
|T4 Ligase | |T4 Ligase | ||
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
