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12. Sample loading buffer: 50% v/v glycerol, 0.25% w/v Bromophenol Blue in
1× TAE.
3. Methods
3.1. Bacterial Fermentation for Plasmid DNA Production
1. Prepare an inoculum of growth media that is 10% v/v that of the final fermentation
volume. Pick a freshly transformed colony of cells and grow the inoculum
overnight (∼16 h) at 37°C and 200 rpm on a shaker incubator in an unbaffled
shake flask. To ensure good aeration, use a shake flask that is at least 2.5 times
the volume of the cell broth (see Note 1).
2. Fill the fermenter vessel with growth medium and autoclave for 30 min at 121°C
(see Note 2).
3. Attach the vessel to a fermenter control unit to maintain the required process
parameters (see Note 3).
4. Once the fermentation culture has cooled to less than 60°C, add 50 μg/ml of
antibiotic (where an antibiotic resistance marker exists), 0.1% v/v polypropylene
glycol (organic antifoam) and 1% w/v glucose aseptically (see Note 4).
5. Set the dissolved oxygen (DO) level (see Note 5).
6. Before adding the inoculum, check that the OD 600 nm reading of the inoculum is
above 1.5 and preferably above 4 before adding to the fermentation vessel (see
Note 6). Add the inoculum when the medium temperature and pH readings obtain
the levels set at step 3.
7. After fermentation is complete (see Note 7), remove the cell broth from the vessel
and harvest the cells by centrifuging at 5000 × g (5300 rpm in a JA-10 centrifuge)
for 10 min at room temperature.
8. A clarified cell lysate can be prepared immediately or the cell pellet can be used
stored at −80°C until further use. Where required, cell pellets can be resuspended
in PBS buffer before storage (i.e., if pellets need to be removed from centrifuge
tubes).
3.2. Preparation of Clarified Cell Lysis
1. Add 3 ml of cell resuspension solution for every 100 ml of pelleted cell culture.
If cells were stored in PBS buffer, centrifuge at 5000 × g (5300 rpm in a
JA-10 centrifuge) for 10 min in a room temperature rotor and then pour off the
supernatant before resuspending in the cell resuspension solution.
2. Add 3 ml of cell lysis solution for every 100 ml of pelleted cell culture and gently
mix by inverting several times. Cell lysis is complete when the solution becomes
clear and viscous (see Note 8).
3. Add 3 ml of neutralization solution for every 100 ml of pelleted cell culture and
gently mix by inverting several times.
4. Centrifuge the solution at 14 000 × g (8900 rpm in a JA-10 centrifuge) for 15
min in a room temperature rotor (see Note 9).