I needed to do a culture and subculture all in one day but my cells are growing slowly. I'm growing BL21-DE3 cells transformed with plasmid and started a culture from a freeze into 3 mL LB+Amp for 6 hours at 37C. I didn't want to lose any of this so I subcultured all 3 mL into 500 mL of LB+Amp at 37C.
I don't think it will be at OD= 0.5 to 0.8 any time soon, but I need to induce it within a few hours. For future reference, will inducing with IPTG overnight at 37C instead of 20C allow the cells to continue growing while still allowing for adequate expression?
Hi, I personally wouldn't induce my cells at 37 °C overnight, the cells grow much faster, will deplete the nutrients sooner and will probably start degrading your protein.
What I usually do is to inoculate cells from -80 °C into fresh medium (5 mL) in the evening and use these grown cells to inoculate large amount of LB (prewarmed to 37 °C) in the next morning. Then it is possible to do induction, cell harvest, lysis and first purification within one day.
You say your cells grow quite slowly, so maybe using pLys strain may help with the growth rate.
I think you should let your starter culture grow overnight and next day shift it into bulk medium. It may help you.
Hi Parisa,
I agree with Jiri and would use an overnight culture to inoculate the next morning to induce at 37 degrees.
If you want to induce overnight I usually inoculate 5ml of medium and then supplement this the next morning with a further 5ml LB + antibiotic to keep the cells happy. As I know it usually takes my cells 2hrs at 37 degrees with pre-warmed LB to reach OD 0.5, I inoculate at 2pm and then start my induction at 4pm and leave overnight between 15 - 20 degrees.
Thanks everyone!
I'm asking more for the case where you didn't forsee needing a particular culture until the day of, and therefore did not start a 5 mL starter culture the night before. It seems from everyone's responses that trying to induce at a higher temperature to mitigate an OD
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