Haynes Lab:Notebook/Engineering PC-TFs/2013/02/01

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2/1/2013

Type IIS Day 3 Assembly

Bacterial transformation

  • Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
  • Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec. (Accidentally for 1 min), and placed on ice.
  • Added 800 μL sterile SOC medium.
  • Grow with shaking at 37°C for 30 min.(Was confused about how to put 2ml tubes in shaker.)
  • Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
  • Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
  • Plate cells on pre-warmed LB agar + antibiotic. (I plated 100 μL of the cells. Protocol didn't specify.) Grow overnight at 37°C.
  • Check results tomorrow.



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