Team:Ionis Paris/06 06 16
To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid. Plasmid DNA : pSB1C3-RFP at 200 pg/µL DH5⍺ competent cells (See 05/06/16) Petri dish LB+Cm: Cm concentration = 25 µg/mL 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 = 0.615 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 23/03.
Transformation efficiency test : Competent DH5⍺ cells with pSB1C3-RFP plasmid
Objectives
Materials
Protocol
