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Team:UNIPV-Pavia/Protocols/Transformation
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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.
