Team:Paris/July 16

Results of transformations

• Positive controls from biobricks 2007, highest number of bacterias than the first try. It's probably due to the highest quantity of bacteria introduced (150µL instead 100µL).

• Transformations from biobricks 2008 don't succeed again.

Preparation of competent cells

• Dilutions 1/200
• Culture during 3h --> DO= 0,15 / 4h --> DO= 0,13
• problem from the spectro, culture have been thrown.

Transformations Biobricks 2008

use of a new protocol --> directly competent cells

• Defroze competent cells on ice during 5'
• Mix 100µL of competent bacterias with 5µL of biobricks 08 (or 1µL for the positive control)
• Incubate 10' on ice
• Heat-shock the cells during 45-50" at 42°C (don't shake!!!)
• Put 2' on ice
• Add 900µL of cold SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Transfer in an eppendorf tube and spin at 5000rpm during 30"
• Remove 800µL of supernatant
• Resuspent the pellet in the 150-200µL left
• Spread on plates
• Incubate O/N at 37°C