Edited by Chang Zhu
1. Inoculate 2 ml LB/antibiotic media with a single colony for each sample.
2. Grow cells overnight at 37 °C with shaking (225-250 rpm).
3. Add 100 ml of the overnight culture to
5 ml LB/antibiotic media.
4. Grow cells for 2-4 hours or until the cells are in mid-log phase
(OD600 = 0.5).
5. Remove 1 ml aliquot of cells to a 1.5 ml eppendorf tube, centrifuge at 12,000 rpm in a mcirocentrifuge
for 30 seconds, remove media, and freeze the cell pellet at -20 °C.
6. Add IPTG to the final concentration of 1 mM, continue incubation
for 2-4 hours at 37 °C with shaking.
7. Remove 1 ml aliquot of cells to a 1.5 ml eppendorf tube, centrifuge at 12,000 rpm in a mcirocentrifuge
for 30 seconds, remove media, and freeze the cell pellet at -20 °C. (Optional: take samples at several time points.)
8. Resuspend cells in 200 ml of 1X Tris-glycine SDS-Polyacrylamide Gel loading buffer.
9. Boil the tubes for 5 minutes, then centrifuge at maximum speed for
5 minutes.
10. Analyze 5-20 ml on a SDS-PAGE gel. Run
samples from the same colony (before and after IGTP induction) side-by-side.
11. After completion of SDS-PAGE gel, staining the gel. Colony expressing
the recombinant protein should show extra bands in the induced samples.
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