Yesterday I did a time course experiment, to see how the 'spontaneous' development of competence in the rich medium sBHI differed between wildtype cells and cells with either the sxy-1 hypercompetence mutation, the purR::kan knockout mutation, or both. But I had to give up halfway through because the cells stopped growing.
Just before I had taken the first samples I had diluted all the cultures in medium from a new bottle, one that had been prepared on a different day, so I wondered if there had been something wrong with this batch, or if I might have forgotten to add one of the needed supplements to it (NAD). I quickly added more NAD to each flask (at time = ~150 minutes), but that didn't boost growth.
I don't think the problem is just how long the bottles of medium had been sitting on the shelf, as the components are typically quite stable. Instead I'm wondering if we might be using medium from a different supplier. In the past I'd noticed substantial differences in how well different brands of BHI supported cell growth, and had sworn to use only the best (Difco), but I know we were recently given some BHI from another supplier. Tomorrow I'll repeat the time course, with the March 1 medium.
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