Team:Paris/July 15

Preparation of Competent Cells

• Dilution 1/200e (1mL of the O/N culture on 200mL of LB without Antiobiotic)
• Culture at 37°C until DO=0,460 (around 3h)
• Sharing into 4 Falcon tubes of 50mL and cooling at 4°C during 40' in ice
• Spin during 8', 2000rpm, 4°C
• Resuspension of the cell's pellet in 20mL of cold CaCl₂ (0,1M) during 30' in ice
• Spin during 8', 2000rpm, 4°C
• Resuspension of the cell's pellet in 1mL of cold CaCl₂ (0,1M)

DNA Extraction of the Biobricks from the registery 2008

• Warming of 10µl of TE at 50°C (in a 1.5ml tube)
• Incubation of "the blotter" (recover by punch tool) during 5min at 50°C in the warm TE
• Incubation 15min at RT (~30°C)
• Recovering of 5µl plasmid

Transformation of the cells

• Mix 150µl of competent cells + 5µl of DNA
• Incubate 30min at 4°C on ice
• Add 200µl of SOC, incubate 1h at 37°C
• Spin 5min, at 2.000rpm, at RT
• Remove 200µl of the supernatant, and resuspend the pellet in 150µl of the supernatant left
• Spread all the suspension on plates containing adaptate antibiotics
• Incubate O/N at 37°C

List of the transformations

• Biobricks 2007

• Biobricks 2008

Culture of Cells

• Culture of DH5ɑ in 10ml of LB without Antibiotics, O/N at 37°C