Hello,

I purchased competent Rosetta2 cells (a BL21 expression strain of E. coli) and my transformations yielded 30-50+ CFUs per SOC plate. However, when I transfer the colonies to liquid culture, there is no growth after 20 hours.

It is a dual antibiotic system, chloramphenicol (100ug/mL) and kanamycin (50ug/mL). I've tried growing the colonies in SOC and LB. However, I am using colonies that I kept at 4C for 72 hours - could this be a problem?

I notice that when I inspect the colonies after refrigeration, there are these crystal growths (my PI believes they are phosphate crystals). Could micro-crystals be forming in my colonies, killing them?

All of my stocks have been autoclaved at 121C for 30 minutes and no one else in lab is having growth issues. I am using filtered tips. Note I am using an expression plasmid that's been successfully used in lab for many years/projects.

My next approach is to try restreaking a colony/repeating the transformation and trying only fresh colonies that haven't been refrigerated. Negative control plates (non-transformed competent cells) don't have any CFUs. Everyone is puzzled at my growth challenges.

Best,

Dan