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