Team:UNIPV-Pavia/Protocols/Transformation

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.