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Contents 1 Lab protocols 1.1 Preparation of chemocompetent DH5-alpha cells 1.2 Retransformation of BioBricks 1.3 Transformation of Ligations (in DH5alpha or XL1Blue)

Lab protocols

On this page you can find a list of important standard protocols we used in our project.

Preparation of chemocompetent DH5-alpha cells

• liquid cultures, OD_600 of 0,6-0,8
• centrifugation at 4 °C, 4500 g, 10 min
• decantation
• resuspend with 40 ml inoune transformation buffer
• repeat centrifugation, decantation and resuspendation
• centrifuge and decantate again
• resuspend in 20 ml inoune transformation buffer
• add 1,5 ml DMSO
• shock freeze in liquid nitrogen

Retransformation of BioBricks

• add 10 µl sterile dest water to DNA on plate
  • incubate for 10 min
  • take 2 µl, leave rest on plate
  • store plates at -20 °C
• add the 2 µl DNA solution to 5 µl competent DH5-alpha
  • incubate for 30 min on ice
  • heat shock for 45 s at 42 °C
  • incubate 3 min on ice
  • add 250 µl LB medium of 37 °C
  • incubate for 45 min at 37 °C, 800 rpm

Transformation of Ligations (in DH5alpha or XL1Blue)

• thaw bacteria on ice or carefully in your hand
• add 2-4µl Ligation mixture to 50µl bacteria
• incubate 30min on ice
• heat shock 30s-45s (XL1Blue preferably 35s) at 42°C
• incubate 6min on ice
• add 250µl prewarmed LB medium (37°C)
• incubate for 45min at 37°C, 800rpm
• plate 300µl on apropiate antibiotic
• dry 15min at room temperature
• incubate at 37°C over night