"
Team:UNIPV-Pavia/Protocols/Transformation
From 2008.igem.org
(Difference between revisions)
| (5 intermediate revisions not shown) | |||
| Line 14: | Line 14: | ||
<br> | <br> | ||
| + | |||
| + | <h1>The protocols we used</h1> | ||
| + | |||
| + | *[[Team:UNIPV-Pavia/Protocols/Lb|LB medium preparation]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Resuspension|Plasmid resuspension from IGEM paper spots]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Transformation|Transformation]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/PlasmidExtraction|Plasmid extraction]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | ||
| + | *[[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/Ligation|Ligation]] | ||
| + | *[[Team:UNIPV-Pavia/Protocols/Pcr|PCR]] | ||
| + | |||
<h1>Transformation</h1> | <h1>Transformation</h1> | ||
| Line 25: | Line 39: | ||
*'''SOC medium''' | *'''SOC medium''' | ||
<br> | <br> | ||
| - | *Put 4-6 µl DNA resuspension into TOP10 tube. | + | *Put 4-6 µl of DNA resuspension into TOP10 tube. |
*Incubate on ice for 30-45 min. | *Incubate on ice for 30-45 min. | ||
*Heat shock: 42°C for 1 min. | *Heat shock: 42°C for 1 min. | ||
Latest revision as of 12:52, 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
- DNA precipitation with sodium acetate
- Antarctic Phosphatase
- Ligation
- PCR
Transformation
(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)
Materials needed:
- LB agar plates with proper antibiotic added incubated at 37°C
- Thawed Invitrogen TOP10 cells (every tube contains approximately 60 µl of competent cells)
- Resuspended DNA
- SOC medium
- Put 4-6 µl of DNA resuspension into TOP10 tube.
- Incubate on ice for 30-45 min.
- Heat shock: 42°C for 1 min.
- Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
- Incubate 2 hours at 37°C, 220 rpm.
- Centrifuge 10 min, 1200 rpm.
- Remove 150 ul of bacteria-free supernatant.
- Plate the remaining part of solution (resuspending the bacteria) on a proper agar plate.
- Incubate overnight at 37°C.
