User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/08/24

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Competent cells


  • Continuation of the elaboration of competent cells.

  • This morning Vlad and I begin with the process of creation of competent cells. We need flasks with LB medium, with the proper antibiotic Nal 20, the first step was to inoculate this flasks with 2 mL of the cultures that Vlad left yesteday. We inoculated 4 flasks with 250 mL of medium and left them incubating at 37°C, 200 rpm.
  • We need this cultures to reach an O.D between 0.45 and 0.55. We performed measures of the O.D with a spectrophotometer an we assure that this O.D was reached. In come cases, because we left the cultures overgrow the O.D was higher but we dilute the cultures with clean LB media and try to reach the proper O.D.
  • Once the cultures where ready we divided all the content in 4 bottles, we do this using a balance and in a flow hood. We centrifugate this bottles for 10 min at 6000 rpm and 4 °C, we eliminate the supernatant and dilute the pellet in 20 mL of the CaCl2 solution that Vlad made yesterday with help of a vortex.
  • We left the bottles on ice and inside a refrigerator all night.