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Bacterial Overexpression Protocol
- Start 5 mL overnight cultures from single colony picks. Grow in shaking incubator at 37C in LB+Amp100.
- Autoclave 200mL LB and a 250mL Erhlenmeyers for each clone. Add Amp to LB. Leave the LB in the non-shaking incubator overnight to get the broth warm.
- The next morning, draw 2 uL of overnight culture and run a PCR to check for the presence of the insert. Use T7 primer and an internal reverse GSP. (Alternatively, do this at the end of the 3-hour grow-out)
- Dilute overnight into 200mL LB+Amp100 and grow at 37C in shaking incubator at 200rpm until OD600 reaches ~0.5 (2-3 hrs from room temp LB).
- Add 500uL 200mM IPTG solution to the 200mL culture.
- After three hours, aliquot samples into labeled 50mL tubes and spin down in swing-rotor at 4C for 20 minutes.
- Store at -80C.
Preparing Bulk Protein for Enzyme Assays
- The DFR assay works well working off protein extracted from the pellets of 100mL of culture grown for 3 hours.
- Thaw pelleted cells on ice. Add 500uL Lysis buffer (to make 10mL of buffer, add to 7.85mL H20: 500uL 1M HEPES, 87mg NaCl, 1mL 200mM PMSF, 100uL 100mM TPCK, 50uL 200mM TLCK) and 10uL Lysozyme (stock: 100mg/mL) to one tube of cells from 50mL of culture. Flick to resuspend pellet.
- Set swing-rotor temperature to 4C.
- Incubate on ice 30 minutes with occasional mixing.
- Spin down 20 minutes at 4C and transfer supernatant to pre-chilled Eppendorf.
- Yield is generally ~1.5mL crude bacterial protein extract from 2 tubes of cells.