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Team:UNIPV-Pavia/Protocols/Ligation
From 2008.igem.org
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*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | ||
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]] | *[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]] | ||
*[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]] | *[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]] | ||
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | ||
| Line 34: | Line 35: | ||
*'''Roche T4 Ligase''' | *'''Roche T4 Ligase''' | ||
| - | *'''Roche T4 Ligase Buffer''' | + | *'''10X Roche T4 Ligase Buffer''' |
*'''ddH2O''' | *'''ddH2O''' | ||
<br> | <br> | ||
*(For every ligation) | *(For every ligation) | ||
| - | + | *Add 50 ng of vector | |
| - | *Add 50 | + | |
*Add [[Image:pv_formula_lig.png]] | *Add [[Image:pv_formula_lig.png]] | ||
| + | *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 | ||
*Add 1 µl of T4 Ligase | *Add 1 µl of T4 Ligase | ||
| + | *10 µl final volume | ||
*Incubate at 16°C overnight | *Incubate at 16°C overnight | ||
<br> | <br> | ||
Latest revision as of 23:06, 1 October 2008
 Home
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|  The Project
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|
|---|---|---|---|---|
 Dry Lab
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|  Modeling
|  Protocols
|  Activity Notebook
|
The protocols we used
- LB medium preparation
- Plasmid resuspension from IGEM paper spots
- Transformation
- Plasmid extraction
- BioBrick digestion with restriction enzymes
- DNA gel extraction
- DNA precipitation with sodium acetate
- Antarctic Phosphatase
- Ligation
- PCR
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.
