Protein Expression and Purification Series - Bio-Rad

CHAPTER 3B
ADVANCE PREP
INSTRUMENTATION PROCES
Protein Expression and Purification Series
Allow the LB agar to cool so that the outside of the flask is just comfortable to hold (around 55°C). A
water bath set at 55°C is useful for this step. Be careful not to let the agar cool so much that it begins
to solidify. Solidified agar can be reheated to melt if antibiotics have not been added.
B. While the agar is cooling, label the plates LB/amp. Label the outside of the lower plate rather than
the lid of the plates that are to be prepared.
C. Ensure LB agar is cooled to 55°C before adding ampicillin since excessive heat will degrade the
ampicillin. Add 200 µl of 50 mg/ml ampicillin to the molten LB agar. The final concentration of the
ampicillin in the LB/amp agar plates will be 50 µg/ml. Swirl or use the stir bar and a stir plate to mix the
ampicillin into the agar taking care not to introduce bubbles.
D. Pour the LB/amp agar plates. One LB/amp plate is required for each team.
Figure 3B.2. Preparation of LB/amp agar plates.
E. After the plates have dried at room temperature for two days they should be stored at 4°C enclosed
in plastic bags or plastic wrap to prevent the plates from drying out. Plates are good for two months.
Pour excess agar in the garbage, not in the sink. Wipe any agar drips off the sides of the plates.
3. Prepare LB broth: At least two days prior to cell culturing, prepare four Erlenmeyer flasks with
100 ml of LB broth: to each of four 500 ml Erlenmeyer flasks, add 100 ml of distilled water and 2
capsules of LB broth. Prepare one Erlenmeyer flask with 200 ml of LB broth: to a 1 L Erlenmeyer
flask, add 200 ml of distilled water and 4 capsules of LB broth. Heat the solutions until the capsules
dissolve. Autoclave all five flasks on wet cycle for 30 minutes. Allow the broth to cool to room
temperature before use.
Note: LB broth without ampicillin is used to rehydrate the BL21(DE3) with pDHFR bacteria.
If an autoclave is not available, LB broth can be filter sterilized through a 0.2 µm filter into a sterile
container, or it can be sterilized in the microwave by heating to boiling at least three times. (Note:
reduce evaporation by using a lower power setting on the microwave so the solution simmers rather
than boils.) Storage of LB broth at 4°C is recommended and can be stored for up to one year. LB
broth can also be stored at room temperature if desired; however, this is not recommended if the
microwave or filter sterilization methods have been used for sterilization, or if the bottle has been
opened after sterilization.
4. Rehydrate bacteria: Using a sterile pipet, rehydrate the lyophilized E. coli BL21(DE3) containing
pDHFR by adding 250 µl of LB broth directly to the vial. Recap the vial and allow the cell suspension
to stand at room temperature for 5 minutes. Store the rehydrated bacteria at 4°C until used (within 24
hours for best results).
5. Set up student workstations according to the student workstation list on page 76.
74 Chapter 3B: Advance Preparation for
Chromatography Instrumentation Protocols