Team:Ionis Paris/23 07 16
The objective is to make a stock of E.Coli DH5⍺ competent cells for subsequent transformations. O/N DH5⍺ pre-culture (made the 22/07): O/N inoculation of 100 µL DH5⍺ into 100 mL LB 0.1M CaCl2: prepared the 23/07 0.1M CaCl2/15% Glycerol: prepared the 23/07 Inoculation of 3 mL O/N culture in 100 mL LB in 500 mL erlenmeyer. Incubation at 250 rpm at 37°C until the DO 0,6 —> DO = 0.591 Cells were transferred to 3 sterile ice-cold 50 mL Falcon tubes. 20 mL in each falcon tube. Incubate on ice for 30 min. Do not allow cells to warm up over 4°C. Spin cells at 4000 rpm for 10 min at 4°C. Discard supernatant and try to drain all remaining media. Gently resuspend on 10 mL cold 0.1M CaCl2 Incubate on ice 20 min Centrifuge 10 min at 4,000 rpm at 4°C Discard supernatant and gently resuspend on 5 mL cold 0.1M CaCl2/25% Glycerol Transfer in 1.5 mL eppendorf (100 µL/tube) Store at -80°C NB: The competency of the prepared cells will be tested the 24/07.
Competent cells: E.Coli DH5⍺
Objectives
Materials
Protocol
Competence:
