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Team:Tuebingen/Notebook/Protocols/ligation
From 2014.igem.org
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<td style="text-align: center">to 10 µL</td> | <td style="text-align: center">to 10 µL</td> | ||
| - | <td> | + | <td>H<sub>2</sub>O</td> |
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| - | <li> Mix all reagents in a PCR-tube and if necessary add to 10 μl with | + | <li> Mix all reagents in a PCR-tube and if necessary add to 10 μl with H<sub>2</sub>O.</li> |
<li> Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.</li> | <li> Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.</li> | ||
Latest revision as of 15:31, 17 October 2014
Protocols
DNA-Ligation
Reagents
| 20-50 ng | linearized vector |
| 1 µL | 10x ligation buffer |
| 1 µL | T4 ligase |
| 1 µL | T4 ligase |
| 1 µL | ATP |
| 100-200 ng | Insert |
| to 10 µL | H2O |
Procedure
- Mix all reagents in a PCR-tube and if necessary add to 10 μl with H2O.
- Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.
- Heat inactivate the enzymes for 5 min at 70 °C.
- Store at -20 °C.
