ic3reyes

Hello, I am trying to overexpress my protein of interest using standard recombinant bacterial expression techniques of batch fermentation in shaker flasks. I have had this problem twice in the past 3 years, where a prep will appear to grow very slowly, then will suddenly flourish 24-48 hours after it is started. The media will turn a yellowish color and smell very strongly of buttered popcorn, and the resulting cell pellet will be much darker in color than the usual E. coli pellet. Oh yeah, and I'll get no protein out of my prep. I'm curious whether anyone knows what common bacteria/fungus/yeast/whatever could possibly get into a culture containing antibiotics and take it over. Has anyone else experienced this phenomenon? Is this something I can prevent in the future in some way? Thank you in advance, Chris Graduate Student University of Arizona

Well, reason #1 is that my PI is a "dogma fanatic", and that's the way they did it when he was a postdoc, so that's the way we do it in our lab . I also think we've had issues with massive gel cracking, maybe due to the low humidity since we're in a desert. But you're right, you don't really need to rehydrate SDS-PAGE gels after destaining before you dry them.

Standard protocol in our lab is to put them in ddH2O for awhile after destaining to let them rehydrate. Doing this before fixing in gel drying film seems to reduce the likelihood of the gel tearing as it dries. I have sometimes left mine in water for up to a week without any damage.