Protein Expression and Purification Series - Bio-Rad

CHAPTER 3A
ADVANCE PREP
CENTRIFUGATION PROCES
Protein Expression and Purification Series
Student Workstations
The following workstation setups are for the initial activities of 1) streaking a starter plate to produce single
bacterial colonies on LB/amp agar plates, 2) preparing overnight cell cultures, and 3) subculturing and
induction of cells and preparing SDS-PAGE samples for later analysis.
1. Streak starter plates to produce single bacterial colonies on agar plates
Each student team requires the following items to streak a starter plate to produce single bacterial colonies:
Material Needed for Each Workstation Quantity
LB/amp plate 1
Sterile inoculating loop 1
2–20 µl adjustable-volume micropipet and tips 1
Marking pen 1
Common Workstation Quantity
Rehydrated BL21(DE3) E. coli containing pDHFR 1
Incubator set to 37°C 1
2. Overnight cell culture
Each student team requires the following items to prepare an overnight culture:
Material Needed for Each Workstation Quantity
LB/amp plate streaked with BL21(DE3) containing pDHFR 1
50 ml conical tube containing 3 ml sterile LB/amp broth 1
20% sterile glucose 300 µl
20–200 µl adjustable-volume micropipet and tips 1
Marking pen 1
Common Workstation Quantity
Tube roller in a 37°C incubator, shaking water bath or shaking incubator set to 37°C 1
3. Subculture and Induction
Each student team requires the following items to prepare a sample of uninduced cells for SDS-PAGE
analysis, to subculture the cells, and to induce the cells:
Material Needed for Each Workstation Quantity
50 ml conical tube containing 3 ml overnight culture 1
50 ml sterile conical tube containing 11 ml sterile LB/amp broth warmed to 37°C 1
LB/amp broth in two 2 ml microcentrifuge tubes for spectroscopy 4 ml
Screwcap microcentrifuge tube, 1.5 ml 1
100 mM IPTG 25 µl
Laemmli sample buffer 1 ml
20–200 µl adjustable-volume micropipet and tips 1
100–1,000 µl adjustable-volume micropipet and tips 1
Semi-microvolume cuvettes 4
Parafilm squares 2
Marking pen 1
60 Chapter 3A: Advance Preparation
for Centrifugation Protocols