Day 3:- To prepare the inoculum for the 2 L flasks, add 5 mL of LB
broth containing 100μg/mL ampicillin to an agar plate that
has grown visible colonies fromstep 10. Using a 10μL loop,
gently lift the cells off the plate. Pipette off the culture and
transfer it to a conical flask labeled pooled inoculum. Repeat
this procedure with a second 5 mL for the same plate. You
should end up with just over 8 mL of inoculum from the plate. - Repeat for each plate. If you had, for example, 12 flasks, you
should end up with around 96 mL of inoculum. - Make up the total volume of the inoculum from 12 using LB
ampicillin according to the following equation: final volume of
inoculum (mL)¼((no. flasks to be grown10)þ10). For
example, for six flasks make up the final volume to 70 mL, or
for 12 flasks make it up to 130 mL. - Grow cultures in 2 L flasks. Distribute 800 mL of LB ampicillin
into each of 122 L flasks and then inoculate each with 10 mL
of inoculum. Ideally the growth should be started at around
1–2 pm to allow cells to be induced and grown overnight. - Grow the bacterial cultures by incubating at 37 C,
210–215 rpm, for 3–4 h or until the OD 600 is 1 (1 cm path
length cuvette). - Induce protein expression in each flask by addition of 7.5 mL
of 100 mM IPTG and incubate for 16–17 h (overnight) at
25 C, shaking at 210–215 rpm.
Day 4:- Harvest the cells: pour cultures from each flask into 1 L centri-
fuge bottles and centrifuge at 4200 rpm (5000g) in a J6-MI
centrifuge for 30 min at 4C. - Wash the cells by removing the supernatant and resuspend the
cell pellet in 20–30 mL lysis buffer (Subheading2.2 above).
Ensure each cell pellet is well suspended by using a homoge-
nizer and then transfer to 50 mL plastic centrifuge tubes. The
supernatant must be carefully disposed of as it contains live
bacteria. Destroy with whatever decontamination process is
required in your laboratory. - Harvest the cell pellet again by centrifuging at 3000 rpm
(1800g) for 30 min at 4C. - Discard the supernatant, weigh the cell pellets, and then freeze
at 80 C until required. Cell pellets are typically 4–5 g of
bacterial pellet per flask.
6 Julia A. Hubbard et al.