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Team:UNIPV-Pavia/Methods Materials/Ligation
From 2009.igem.org
(Difference between revisions)
| Line 11: | Line 11: | ||
*(For every ligation) | *(For every ligation) | ||
*Add 50 ng of vector | *Add 50 ng of vector | ||
| - | *Add | + | *Add <html> <img src="https://static.igem.org/mediawiki/2008/8/82/Pv_formula_lig.png"></img></html> |
*Heat DNA mix at 65°C for 5 min for DNA denaturation | *Heat DNA mix at 65°C for 5 min for DNA denaturation | ||
*Add 1 µl of T4 Ligase buffer | *Add 1 µl of T4 Ligase buffer | ||
Revision as of 17:25, 22 June 2009
Ligation
(estimated time: 20 min + 12-16 hours overnight incubation)
Materials needed:
- Roche T4 Ligase
- 10X Roche T4 Ligase Buffer
- ddH2O
- (For every ligation)
- Add 50 ng of vector
- Add
- Heat DNA mix at 65°C for 5 min for DNA denaturation
- Add 1 µl of T4 Ligase buffer
- Add 1 µl of T4 Ligase
- 10 µl final volume
- Incubate at 16°C overnight
- Then, ligation can be conserved at 4°C or can be transformed
- Before transformation you have to inactivate T4 Ligase:
- Heat ligation at 65°C for 10 min.
