User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/04/13

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Objective

Transform the mutated GFP into an expression strain so it can be re-expressed for purification.

Description

Transformation

  1. Mix 20μL of BL21(DE3) Competent E.coli with 5μL of the mutated GFP (D1C) in the cold, sterile tube.
  2. Incubate this mixture for 30 minutes on ice.
  3. Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
  4. Add 250μL of SOC media to the cells/PCR product.
  5. Shake the mixture at 250rpm at 37°C for one hour.
  6. 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.
  7. Incubate the plate overnight (inverted) at 37°C.

Notes

Always make glycerol stocks after a transformation is completed, otherwise if something goes wrong you'll have to do the transformation again.


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