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Team:UNIPV-Pavia/Protocols/Ligation
From 2008.igem.org
(Difference between revisions)
| Line 40: | Line 40: | ||
*Heat vector at 65°C for 5 min for DNA denaturation | *Heat vector at 65°C for 5 min for DNA denaturation | ||
*Add 50 µg of vector | *Add 50 µg of vector | ||
| - | *Add <math>6*50*length(insert)/length(vector)</math> | + | *Add <math>\6*50*length(insert)\/length(vector)</math> |
*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 | ||
Revision as of 12:44, 1 July 2008
 Home
|  The Team
|  The Project
|  Biological Safety
|  Parts Submitted to the Registry
|
|---|---|---|---|---|
 Dry Lab
|  Wet Lab
|  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
- Antarctic Phosphatase
- Ligation
- PCR
Ligation
(estimated time: 20 min + 12-16 hours overnight incubation)
Materials needed:
- Roche T4 Ligase
- Roche T4 Ligase Buffer
- ddH2O
- (For every ligation)
- Heat vector at 65°C for 5 min for DNA denaturation
- Add 50 µg of vector
- Add <math>\6*50*length(insert)\/length(vector)</math>
- Add 1 µl of T4 Ligase buffer
- Add 1 µl of T4 Ligase
- 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.
