Overexpression test

DNA for gene of interest has already been transformed into a cell type optimized for protein production from a plasamid (BL21 DE3, Rosetta)
Recommended to test 4 colonies

Day 1

1. Prepare overnight culture of 5 mL LB + 5 μL antibiotic(s)
2. Inoculate with one colony from plate
3. Grow at 37�C with shaking for 12- 16 hours

Day 2

1. Obtain sterile flasks with 30 mL LB
2. Add 30 µL antibiotic and 300 µL O/N culture
3. Incubate cells 37�C
4. Make glycerol stocks from overnight cultures: Add 150 μL sterile 100% glycerol and 850 μL of O/N culture to cryo vial, store at -80�C
5. Let cells grow until OD 600nm is between 0.6 and 0.8 (about 3 hours)
6. Take gel sample: Spin down 0.5 mL cells, decant sup. Resuspend pellet in 100 μL H2O, add 34 μL of 4X SDS
7. Induce cells to overexpress protein with 0.4 mM IPTG (for 28 mL, add 11.2 µL of 1 M IPTG)
8. Put in shaker at 37�C for 4 hours
9. Take gel sample after 4 hours, bleach and discard culture, run gel to analyze overexpression