User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/21

PcTF Subcloning in E. coli

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01/21/2013 Chemically Competent Cell Prep (BL21-DE3) Haynes Lab, 2013 Prepare these solutions ahead of time and chill at 4°C overnight: 100 mM CaCl2, autoclaved 15% glycerol in sterile 100 mM CaCl2 First autoclave the 100mM CaCl2 and then make 15% glycerol solution. (Do not autoclave the glycerol) Grow untransformed cells on plain LB (streak for single colonies). Inoculate 12.5 mL culture with colony from plate. Incubate with shaking at 37°C for 8 hours. Prepare two 250 mL of plane LB Broth and autoclave. Transfer 6.2 mL culture into 250 mL. Grow until absorbance of 0.6 A600 is reached. BL21 growth is slow, overnight incubation is recommended. (12hrs) Aliquot 125 mL culture to 4 sterile large 250mL polycarbonate spin bottles. Chill on ice for 15 min. Centrifuge cells at 4000 rpm for 10 min ate 4°C. (Dr. Wang's lab refrigerator centrifuge) Resuspend cells in sterile, ice-cold 100 mM CaCl2 by gentle pipetting (do not vortex!). Use 37.5 mL per 125mL culture (75 mL per 250 cell culture). Incubate on ice for 15 min. Centrifuge cells at 4000 rpm for 10 min at 4°C. Resuspend in sterile, ice-cold 100 mM CaCl2/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture. The cell pellets are very sticky. We need to pipette up and down for a long time to get them completely resuspended. Leave on ice in a cold room/ fridge for 21 hours. Freeze 200μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C.