Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Chapter 4: Culturing, Expression, Lysis and SDS-PAGE
Analysis for 11 ml Cultures for Centrifugation Purification
Process
Cell Culture and Induction
Student Workstations
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
Each lab team will streak their own starter plate as a source of cells for culture for protein production.
LB/amp plates should be streaked for single colonies and incubated at 37°C for 16–24 hours before the
initial culture activity is planned.
Student Protocol: Streaking starter plates to produce single bacterial colonies
on agar plates
Using the rehydrated BL21(DE3) E. coli containing pDHFR at the Common Workstation to streak
one plate. The purpose of streaking is to generate single colonies from a concentrated suspension of
bacteria. A minute amount of the bacterial suspension goes a long way. Under favorable conditions,
one cell multiplies to become millions of genetically identical cells in just 24 hours. There are millions of
individual bacteria in a single 1 mm bacterial colony.
1. Pipet 10 µl of reconstituted E. coli using a sterile pipet tip onto an LB/amp plate. Use a sterile loop to
perform the streaking. Streaking takes place sequentially in four quadrants. The first streak is to just
spread the cells out a little. Go back and forth with the loop about a dozen times in one quadrant as
shown in Figure 4.1a. In subsequent quadrants the cells become more and more dilute, increasing the
likelihood of producing single colonies.
2.
For subsequent streaks, the goal is to use as much of the surface area of the plate as possible. Rotate
the plate approximately 45 degrees (so that the streaking motion is comfortable for your hand) and
start the second streak. Do not dip into the rehydrated bacteria a second time. Go into the previous
streak about two times and then back and forth as shown in Figure 4.1b for a total of about 10 times.
3. Rotate the plate again and repeat streaking (Figure 4.1c).
Rotate the plate for the final time and make the final streak (Figure
4. 4.1d). When you are finished
streaking the plate, cover it immediately to avoid contamination. Label the plate with your initials.
Chapter 4: 11 ml Culture Protocol for
Centrifugation Purification
91
CHAPTER 4
11 ml CULTURE
PROTOCOL