User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/03/26

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Transformation and Preparation for Expression

Transformation of M33S/M103S into Expression Strain

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 40μL of BL21(DE3) E.coli with 5μL of the M33S/M103S mini-prep (col. 1) 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.

Preparation for Expression

  • 1L LB = 25g LB + 1L distilled H2O (combine in 2800mL flask, autoclave on liquid cycle)
    • four of these were prepared
  • 25mL LB = 0.625g LB + 25mL distilled H2O (combine in 250mL flask, autoclave on liquid cycle)
    • four of these were prepared