"
Team:UNIPV-Pavia/Methods Materials/Transformation
From 2009.igem.org
(Difference between revisions)
Susanna (Talk | contribs)
(New page: {{UNIPV-Pavia/Protocols}} <br> == '''Transformation''' == ''(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)'' <br> <br> '''Materials needed:''' *'''LB agar plates ...)
Newer edit →
(New page: {{UNIPV-Pavia/Protocols}} <br> == '''Transformation''' == ''(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)'' <br> <br> '''Materials needed:''' *'''LB agar plates ...)
Newer edit →
Revision as of 16:40, 22 June 2009
- LB medium
- DNA resuspension from iGEM 2009 plates
- Transformation
- X-Gal staining protocol for beta galactosidase (blue/white screening)
- Glycerol stocks
- Plasmid extraction
- BioBrick digestion with restriction enzymes
- Electrophoresis
- DNA gel extraction
- DNA precipitation with sodium acetate
- Ligation
- PCR
- Sensing ethanol concentration: our do-it-yourself kit
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.
