User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/04/09

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Transformation of Triple Mutant into Expression Strain and Sonication of Double Mutant Expressed Cells

Transformation of M33S/M103S/M8S into Expression Strain

  1. Take a sterile eppendorf tube and place it on ice.
  2. Mix 30μL of BL21(DE3) E.coli with 5μL of the M33S/M103S/M8S mini-prep (col. 3-sequence confirmed) 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.

Sonicate M33S/M103S BL21(DE3) E.coli to extract protein

  1. Safety First: Make sure to wear noise blocking earmuffs.
  2. Sonicate each tube for 30 seconds at power setting 11.
  3. Place each tube on ice for at least 30 seconds after sonication.
  4. Repeat this procedure two more times.
  5. Distribute sonicated cells between 6 centrifuge tube for a balanced spin.
  6. Centrifuge the cells for 2 hours at 4°C at 18000rpm.
  7. Pour off and store supernatent at 4°C.