Day 1 : 06/06/2017

Cleaning the laboratory, then recovery and installation of the equipment. Starting culture of strains TG1 and DH5α. We launched precultures from cryotubes ( sampling with 1 rod and then deposition in an erlenmeyer containing LB media ). Incubation at 37 ° C.

Day 2 : 07/06/2017

Strains cultivation: Recovery of strains cultured the day before: DH5α and TG1 To check the concentrations, the OD is measured. For this purpose, the culture must be diluted because the optimum measurement of the apparatus is between 0.1 and 0.8. The cultures in the tank are diluted to 1/10 with distilled water. Therefore, TG1: 5.16 / DH5α: 5.21 To calculate the volume to be taken for our 100 mL we do: $$\frac{\text{DO voulue}}{\text{DO obtenue avant dilution}} \times \text{Volume} = \frac{0.1}{5.16} \times 100 \simeq 2 \text{ mL}$$   E. coli being aerobic, the solution is stored in LB in a Erlenmeyer flask of 5 × volume, so 500 mL and incubate for 1 h.

Glycerolization of strains:   Putting the strains in glycerol protect them from the cold, necessary when one will freeze at -80 ° C so that the cells do not collapse. 40% Glycerol in 1.8mL cryotubes → 0.9 of glycerol and 0.9 of strains. We will freeze the cells in exponential phases in order to make our cells competent, ie able to incorporate DNA.

Competent bacteria: To make competent bacteria, it is necessary to take them in exponential growth phase. A quantity of bacterium is thus taken which is placed in LB medium and then incubated for 1 hour at 37 degree Celsius. An OD value of 0.1 is desired in 100 mL. For TG1-> 1.94 mL For DH5α-> 1.92 mL