From 2008.igem.org

Latest revision as of 12:52, 1 July 2008

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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.

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