I have been trying to grow Rosetta 2 Cells transformed with Tri-Ex4 DHX36 plasmid in 600 mL culture. I transform the Rosetta 2 competent cells and plate them selected with Chloramphenicol 35ug/mL and Ampicillin 50ug/mL. The cells transform but give me two different size colonies after 24 HR incubation at 37C. One is slightly larger but few in number, and the smaller ones are more in number. I take both types of colonies and grow them separately in a 3 mL LB starter culture with the same antibiotics overnight. The next day, I tried to grow them in a larger 600 mL culture with the same antibiotics to grow them to 0.6 OD for IPTG induction for protein expression. However, even with a 1:1000 transfer (600 uL of starter culture in 600 mL LB media) or dumping the whole 3 mL culture in 600 mL, it shows almost no growth in 6 hours. If I end up leaving it overnight, the cells grow and saturate the media, and at that point, I have no way of doing IPTG induction. (Sometimes, I start to get OD 0.1 after 6 hours of incubation.)