Protein expression

• Inoculate media (+ antibiotic) with transformed cells from a fresh plate (<1 week old) to form a pre-culture. For protein expression, a single colony is not necessary. Use 10ml pre-culture per 1l final culture volume and grow in tubes giving plenty of room for aeration (ie a max of 10ml in 1x50ml falcon tube).

• Incubate pre-culture overnight at 37^oC. In theory, cultures should not be grown for >16 hrs.

• Use pre-culture to inoculate growth media (+antibiotic). There should be 1l of autoclaved culture in a 2l Erlenmeyer flask. Grow at 37^oC.

• When OD₆₀₀ is 0.4-0.8 (ie mid-log phase), induce cells with IPTG (final concentration 400-1000uM). Flasks inoculated with 10ml pre-culture at 10am should be ready to incude ~1-2pm. Remember that you can calculate the rough time needed to reach the correct OD from an earlier measurement: OD doubles ~every 30mins.

• Grow cells overnight at 21^oC before harvesting.

Parameters that may need to be optimised dependent on protein are OD of induction, concentration of IPTG (poss), length of growth after induction, growth temperature after induction.

To harvest cells, centrifuge cell cultures at 5000rpm for 10-15 mins. Discard supernatent as biohazard waste (decontaminate with virkon prior to putting down sink).