User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/02/27

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Transformation of wt swMb pT7-7 (Myoglobin)

  1. Remove wt swMb pT7-7 from freezer, thaw, and let sit at room temp for about a half hour.
  2. Take a sterile eppendorf tube and place it on ice.
  3. Mix 50μL of NovaBlue Competent E.coli with 5μL of the wt swMb pT7-7 in the cold, sterile tube.
  4. Incubate this mixture for 30 minutes on ice.
  5. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  6. Add 200μL of SOC media to the cells/PCR product.
  7. Shake the mixture at 250rpm at 37°C for one hour.
  8. Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plate was made with 0.875g LB, 0.7g Agar, and 35mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35μL of 100mg/mL ampicillin was added to it and it was poured into a sterile petri dish.
  9. Incubate the plate overnight (inverted) at 37°C.

Update 2/28/12: 11 colonies grew overnight on the plate