Protein Expression and Purification Series - Bio-Rad

CHAPTER 4
11 ml CULTURE
PROTOCOL
Protein Expression and Purification Series
Measure OD600 of subculture and induce GST-DHFR-His expression
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After one hour of incubation measure the OD600 of your subculture.
Blank a spectrophotometer at 600 nm with 1 ml LB/amp broth.
Prepare a 1:3 dilution of your subculture by combining 600 µl of sterile LB/amp broth with 300 µl of
subculture in a clean semi-microvolume cuvette. Cover the cuvette with parafilm and invert gently
several times to mix well.
Measure the OD600 of your 1:3 diluted subcultured cells and record the results below. Next calculate
the OD600 of the subcultured cells and record the value.
OD600 of 1:3 dilution of subcultured cells: ________________
OD600 of subcultured cells = OD600 of 1:3 dilution x 3: ________________
Add 25 µl of 100 mM IPTG to the subculture in the 50 ml conical tube and continue incubating and
shaking the culture at 37ºC for 4–24 hours. During this time period, most of the resources of the E. coli
will go toward producing the recombinant GST-DHFR-His protein rather than cell division, though cell
division does still occur.
Record the time that you start and end your induction below.
Induction Start Time: _______________________________
Induction End Time: ________________________________
96 Chapter 4: 11 ml Culture Protocol
for Centrifugation Purification