Team:TU Darmstadt/Protocols/Metabolism

Protocols Metabolism

The following page(s) give an overview of the standard protocols used by the team Metabolism in iGEM PET.erminators project 2012.

In vivo

Production of chemically competent cells

Production of chemically competent cells

• Inoculate 10 ml of LB-Media with an E. coli colony and incubate at 37 °C overnight
• Inoculate 200 ml of LB-Media with 2.5 ml of the overnight culture
• Incubate the culture at 37 °C until an OD600 of 0.5
• Cool the cells on ice for 5 min
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the cell-pellet in 32 ml pre-cooled CaCl₂ solution (Do not vortex!)
• Add pre-cooled CaCl₂ solution to 200 ml
• Let the cells repose on ice for 1 hour
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the pellet in 10 ml cryo-solution
• Decant 200 µl of competent cells in a 1.5 ml tube
• Store the tube in an -80 °C freezer

Solutions

• CaCl₂
  • 5.55 g CaCl₂
  • Add di H_{2>/sub>O to 1 L}
  • Sterilize by autoclaving

• Cryo solution
  • 0.278 g CaCl₂
  • 10 ml glycerin
  • Add di H_{2>/sub>O to 50 ml}
  • Sterilize by autoclave