I was wondering if you have tried this protocol with BL21DE3 RIPL cells. Is the protein expression from the stock culture as good as from fresh ones?
Make a Stock Culture
The way to work with BL21 clones is to make stock cultures from fresh transformants and use these stock cultures for the expression work. This insures that the clone does not change and that each expression run gives optimal performance.
1. Transform the BL21 strain to be used with your plasmid (usually 1uL of a 1:50 dilution is enough).
2. Pick a single transformant colony from a fresh plate into 30 mL of LB + antibiotic.
3. Grow overnight. Room temperature is best. Turn the heater off on the shaker. Don't worry, the cells will grow. Many people cannot just shut the heat off on a shared lab shaker. In that case, grow the cells at 30°C. If a 30°C shaker isn't available and they have to grow at 37°C.
4. In the morning, dilute 10 mL of the overnight with 10 mL of LB-20% glycerol. Note OD.
5. Distribute 1 mL each into 1.2 mL cryotubes (5 to 20 tubes). Freeze and store at -70°C. These are stocks. As long as they stay at -70°C, they will be unchanged.
6. Each time you do an expression, thaw out a stock culture and use that to start the culture.
Growth and Sampling
1. Dilute 1 mL of stock culture into 100 mL of media + antibiotics in a 500 mL baffled flask.
2. Grow the cells to 0.5 OD at 37°C. This should take about 2-3 hours. During this time, mark 5 conical 15 mL centrifuge tubes: 0, 30 min, 2 hrs, 4 hrs, O/N.
3. Harvest a 10 mL sample of the uninduced (0 hours) sample. Spin the tube at 4,000 rpm for 20 minutes. Pour off the supernatant. Freeze the pellets.
4. Add 1 mL of 100 mM IPTG to the culture [final conc. 10 mM].
5. Measure the OD of the cells for each of the next 3 hours. Harvest 10 mL samples of the culture each time: 30 min, 2 hrs, 4 hours after induction. Store the pellets at -20°C.
6. Continue to express the cells overnight.
7. The next morning, harvest 10 mL of the cells. Note the time.
Hi Natashcha, I think that this protocol is OK. But with protein expression every case is unique and special. I expressed a lot of proteins using frozen stock culture and I had a construct which could be expressed only from the freshly transformed cells. Usually I make stocks from overnight culture by adding 150-200 ul of glycerol directly to 1 000ul of o/n culture .I freeze culture on dry ice- EtOH bath and keep at -80C. For the expression experiments I scoop some frozen culture by sterile tip and inoculate 50-100 ml flask with media and antibiotics, incubate overnight and use this culture to inoculate 500 ml-1000 ml flasks. To have a good expression you need to start with “happy “cells. I recommend you to make a stock and try small scale expression form frozen cells first. Good luck with your experiments!
Tetyana
- Badges
- Science topic