Transform the mutated GFP into an expression strain so it can be re-expressed for purification.
- Mix 20μL of BL21(DE3) Competent E.coli with 5μL of the mutated GFP (D1C) in the cold, sterile tube.
- Incubate this mixture for 30 minutes on ice.
- Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
- Add 250μL of SOC media to the cells/PCR product.
- Shake the mixture at 250rpm at 37°C for one hour.
- 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.
- Incubate the plate overnight (inverted) at 37°C.
Always make glycerol stocks after a transformation is completed, otherwise if something goes wrong you'll have to do the transformation again.