Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Overnight Cell Culture
1. Prepare LB/amp broth: Ensure LB broth is cooled to room temperature before adding ampicillin;
excessive heat will degrade the antibiotic. Add 100 µl of 50 mg/ml ampicillin to each of the four
Erlenmeyer flasks containing 100 ml of LB broth. Swirl to mix. Add 200 µl of 50 mg/ml ampicillin to the
200 ml of LB broth in the 1 L Erlenmeyer flask and swirl to mix. The final concentration of ampicillin in
the LB broth in all bottles and flasks will be 50 µg/ml.
* LB broth containing antibiotics should be stored at 4°C and is good for one month.
2. Dispense LB/amp broth into sterile 50 ml conical tubes: Using the LB/amp broth from the 1 L
Erlenmeyer flask, dispense 15 ml of LB/amp broth into one 50 ml sterile conical tube per workstation.
Dispense 2 ml of LB/amp broth into 2 ml microcentrifuge tubes, one per workstation. Store at 4°C
until ready for use.
3. Aliquot 1 ml of 20% sterile glucose into a 2 ml microcentrifuge tube for each workstation.
4. Set up student workstations according to the student workstation list on page 76.
Subculture and Induction
1. Refer to pages 26-30 to help determine the timeline for induction that the class will use. A four hour
induction provides optimal results.
2. Make sure that the incubator shaker or shaking water bath is set to 37°C.
3. Warm up Erlenmeyer flasks containing 100 ml LB/amp broth: At least one hour before the
laboratory period, place the four 500 ml Erlenmeyer flasks in a 37°C incubator shaker, or shaking
water bath and warm the medium to 37°C.
4. Set the shaking speed of the shaking water bath or incubator shaker to 250–275 rpm. Once the cells
are added, it is essential that the cells remain suspended and oxygenated for proper growth. There
should be some foaming when the cells are shaken.
5. Turn on the spectrophotometer and allow it to warm up for at least 30 minutes. If using the
Bio-Rad SmartSpec Plus spectrophotometer, step-by-step instructions are available in Appendix
D for setting up the instrument for reading absorbance at 600 nm. If using another manufacturer’s
spectrophotometer, please consult the instruction manual for that instrument for setting it up to
measure absorbance at 600 nm.
6. Aliquot 25 ml of 1 M IPTG into a 2 ml microcentrifuge tube for each workstation.
7. Aliquot 1 ml of Laemmli sample buffer into a 2 ml microcentrifuge tube for each workstation.
Note: The set up as described involves distributing 1 ml of Laemmli sample buffer at the subculture
and induction stage for each student workstation. This sample buffer will be used throughout the
protocols and should be saved by the students. Alternatively, aliquots can be distributed each time the
Laemmli sample buffer will be used.
8. Set up student workstations according to the student workstation list on page 77.
Chapter 3B: Advance Preparation for
Chromatography Instrumentation Protocols
75
CHAPTER 3B
ADVANCE PREP
INSTRUMENTATION PROCES