Team:Paris/August 1

souche stockage

MiniPreps

• Use of Quiagen's protocol on all the clones cultivated on the 31th.
• Preparation of 50µl of minipreps in water.

List of Minipreps

Transformations

Protocol

use of TOP10 Chemically competent cells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspent the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

List of transformation