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Team:UNIPV-Pavia/Protocols/Ligation
From 2008.igem.org
(Difference between revisions)
| Line 35: | Line 35: | ||
*'''Roche T4 Ligase''' | *'''Roche T4 Ligase''' | ||
| - | *'''Roche T4 Ligase Buffer''' | + | *'''10X Roche T4 Ligase Buffer''' |
*'''ddH2O''' | *'''ddH2O''' | ||
<br> | <br> | ||
Latest revision as of 23:06, 1 October 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
- 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.
