Manish, I also had a similar problem getting cells (C41 (DE3) in my case) to grow in minimal media. To get around this problem, I took cells from an agar plate and grew them in a small volume (5 mL) of the minimal media. Once that culture got thick, I then inoculated 200 mL of minimal media with 0.5 mL of the 5 mL culture. I would let this grow, and I used this as my overnight culture to inoculate my large flasks. Long story short, the cells seemed happier adjusting to the minimal media in a smaller volume first.
Cheers, Jamie Wallen > Hello everybody, > > Sorry for an offtopic question. I am trying to express a protein in M9 > minimal media for Selenomet incorporation. When grown in LB this protein > expressed very well and got good crystals. Diffraction was upto 2 A. I am > having a hard time expressing the same protein in Minimal media. It took > nearly 24 hours for the OD600 to reach ~ 0.4 before inducing with IPTG in > minimal media and eventually got no protein expression. It looks like the > cells are not growing or taking very long to grow. The cell line I am > using is RossettaBlue (DE3) and the plasmid is pET19b based (Novagen). > > It expressed poorly in BL21 (DE3) when grown in LB and thus decided to use > RosettaBlue (DE3). It worked very well in LB, but having a hard time > while expresing the same in minimal media using Rosetta Blue. Has anybody > tried expression in minimal media using Rosetta Blue cell line? I am > planning to try overnight induction. > > Any suggestions would be greatly appreciated. > > Thanks, > > Manish > > > > > > > > > ************************************************* > Manish B. Shah, PhD. > Postdoctoral Fellow > Hauptman-Woodward Medical Research Institute > 700 Ellicott Street > Buffalo, NY 14203. > ************************************************* >
