1. Cells for new DNA preps: For the replicate DNA preps (for the replicate LC-MS analysis), yesterday I inoculated GFAJ-1 cells into two 50 ml cultures in AML60 medium with 1500 µM PO4, with and without 40 mM AsO4, and into two 500 ml cultures on AML60 medium with 3 µM PO4, with and without 40 mM AsO4. Most of these cultures are growing nicely, so tomorrow I think I'll have enough cells for the DNA preps. Well, the 1500 µMp 40 mM As culture isn't growing at all, but I don't think we need to replicate this one anyway. I need to get at least 50 µg of DNA from each prep, to give the grad student enough for his CsCl gradients. Last time one of the cultures (3 µM PO4, no AsO4) wasn't dense enough to give me the DNA I needed, but so far it looks as dense (or not-dense) as the parallel culture with AsO4. I'll prep the DNA today and if I don't have enough I'll just set up more cultures. I'd be able to prep the DNA from them on Sunday, so still would have the DNAs ready to send on Monday.
2. Troll-suggested control: I've run the gel of the two-month-old DNAs from cells growth with and without arsenic, both native and denatured, and there's no difference in fragment length, with all double-stranded fragments being at least 30 kb in length. So there's no evidence of arsenic-bond strand breakage during long-term storage at 4 °C. I'll post a gel photo later (the image I saved isn't right).
3. Presentable growth curves: A lab in our research cluster has a BioScreen incubator/plate reader I can use to automate my growth curves. But the test cultures I set up in an ordinary microtiter plate aren't growing consistently, so I'll have to mess around a bit before I can do the growth curves.
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Thanks so much for keeping us in the loop with the #arseniclife story. It's been a fascinating read over the last year as well as your detailed thoughts over your own research topics!
ReplyDeletedid we ever get to the bottom of why felisa's +as dna preps migrated differently from her -as in the agarose gel (ignoring the rRNA contamination) whereas your +/- as preps looked identical? is it in any way possible that her dna in the +as contained arsenic and had undergone some hydrolysis and thus was resembling your hypothetical agarose gel experiments you posed a few posts back in response to the 'troll' — just seems like there are some differences between her preps and yours, and wouldn't want those differences to be the difference between detecting As and not detecting it
ReplyDeleteI think the W-S et al. DNA preps migrated differently because the +P lane had so much more RNA (and probably DNA) than the -P lane. The RNA in the +P lane had swept up all the EthBr in its path, so the DNA is only stained at the edges of the lane and has the streakiness that's characteristic of overloaded high-molecular-weight DNA. There could also have been differences in how much salt was in the different preps, as they had only been partially purified.
ReplyDeletep.s. The gel in this post is the experiment that tests the troll's concern.