Transform the mutated GFP into an expression strain of E. coli. Prepare the amylose resin for purification. Prepare media for E.coli expression.
- Mix 30μL of BL21(DE3) Competent E.coli with 5μL of the mutated GFP 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.
Regeneration of Amylose Resin Column
- Run 90mL of distilled water over the column at 4mL/min.
- Run 90mL of 0.1% SDS over the column at 4mL/min.
- Run 30mL of distilled water over the column at 4mL/min.
- Run 150mL of column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) over the column at 4mL/min.
Preparation of Media for Expression
- Combine 25g LB and 1L of distilled water in a 2.8L flask. (2 flasks)
- Autoclave the flasks (liquid cycle).
- The plate that was incubated overnight (from the GFP transformation) grew many colonies. The colonies had a light green color.