Protein Expression Protocol

When clone is verified by sequencing, transform the reaction into competent cells used for protein expression (ex: BL21). After transformation is grown over night in 5mls luria broth (LB) with appropriate antibiotic concentration of 50g/mL, spin down culture into pellet, remove 4mLs and resuspend pellet in remaining 1mL. Add 350mLs glycerol to resuspended pellet and mix until mixture is homogenous. This is your stock of cells for protein expression. For expression: Use 1/10 of glycerol stock ~35L to 30mLs LB with appropriate antibiotic concentration of 50g/mL. *** keep track of volume for later *** growth cultures in a 37C shaker 1. Growth 1mL o/n culture in 20mL fresh LB with 20L antibiotic until O.D reaches 0.5. Do this by removing 1mL of culture and placing it in cuvettes, using LB as your blank. 2. When O.D reaches 0.5 to 0.6 remove 1mL, spin it down, remove supernatant and resuspend pellet in 100l of 2x solubilization buffer . Boil sample for 5 minutes. This is your uninduced sample. 3. Add your induction reagent. 4. Remove 1mL of culture every hour for 4 or 5 hours, spin down and resuspend in 100l of 2x solubilization buffer, boil for 5 minutes. 5. Using a rainbow protein ladder, run your samples on a protein gel.