TSS competent cell preparation

1) Inoculate a culture from an ovenight culture at 1/50. 2) Grow until OD600 ~ 0.5-0.6. 3) Cool the culture in an ice/waterbath slurry. 4) Spin the bacteria at 4°C for ...
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Microbiology

TSS competent cell preparation

Before starting, put centrifugation bottles, Falcon tubes and ddH2O and or 10% Glycerol at 4°C. Also precool the centrifuges.

1) Inoculate a culture from an ovenight culture at 1/50. 2) Grow until OD600 ~ 0.5-0.6 3) Cool the culture in an ice/waterbath slurry 4) Spin the bacteria at 4°C for 10 minutes at 3500 rpm. 5) Resuspend the cells in 1/10 of original volume of TSS. 6) For transformation, heat shock the cells for 45 seconds at 42°C.

TSS Buffer: 4.5 ml 10% Polyethylene glycol 0.5 g 5% Dimethyl Sulfoxide 0.25 ml 20mM MgCl2 0.1 ml 1M Bring to 4.5 ml with autoclaved LB

50 ml 5g 2.5 ml 1 ml

Filter through 0.22 µM syringe filter. Store at -20°C

Microbiology 6