Team:Paris/August 1

Conditions

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

Protocol

 * 1mL glycerol
 * 1mL culture in LB

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