User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/03/09

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Transformation of wt swMb pT7-7 (Myoglobin)

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 30μL of BL21(DE3) E.coli with 5μL of the wt swMb pT7-7 in the cold, sterile tube.
  3. Incubate this mixture for 30 minutes on ice.
  4. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  5. Add 200μL of SOC media to the cells/PCR product.
  6. Shake the mixture at 250rpm at 37°C for one hour.
  7. 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.
  8. Incubate the plate overnight (inverted) at 37°C.


Transformation of PCR Product from 3/7/12

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 40μL of BL21(DE3) E.coli with 5μL of the PCR product in the cold, sterile tube.
  3. Incubate this mixture for 30 minutes on ice.
  4. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  5. Add 200μL of SOC media to the cells/PCR product.
  6. Shake the mixture at 250rpm at 37°C for one hour.
  7. 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.
  8. Incubate the plate overnight (inverted) at 37°C.

Making Media

  • 50mL LB = 1.25g LB + 50mL H2O (autoclave)
  • 100mL LB = 2.5g LB + 100mL H2O (autoclave)