For credible DNA preps I need reproducible growth with and without 40 mM arsenate, and I need to be able to grow large volumes because the cultures with only 3 µM added phosphate won't reach a very high density and thus won't yield much DNA per ml.
One practical limitation to my GFAJ-1 experiments is that it's optimum growth temperature is 28 °C but we only have two shaking water-baths, one that's always at 37 °C and one that currently often needed at 30 °C. But Halomonas bacteria typically tolerate a wide range of growth temperatures, and there's 30 °C room down the hall with shakers in it. So tonight I'm setting up pairs of replicate cultures without arsenate, one to shake at 28 °C and one to shake at 30 °C. If they grow at about the same rate and to the same density I'll do my growth tests in the 30 °C room and only switch to our own shaking water-bath at 28 °C when the arsenate-resistance problem has been sorted out and I'm ready to generate good growth curves and DNA preps.
So... Make up a fresh batch of medium, add cells from a freshly thawed tube of GFAJ-1, and put into replicate flasks with different amounts of phosphate. Do I have enough glass flasks? Yes, there are 12 on the sterile-glassware shelf.
_(cropped).jpg)
3 weeks ago in Genomics, Medicine, and Pseudoscience
could the plastic tube/glass tube growth discrepancy have to do with how much oxygen is getting in there? if one is an airtight seal and the other is not...
ReplyDeleteBoth seal pretty tight, though the plastic may be more permeable to both O2 and CO2. Another reader suggested that the CO2 might be affecting pH, but all the tubes had very similar pHs.
ReplyDeleteHello,
ReplyDeleteIn previous experiment it was proved that, the cells grew well with arsenate in small glass screw-cap tubes , now soaking in preparation for future re-use, but my DNA preps will need much larger volumes ,500 ml or more. Because cells can't grow to high density when phosphate is limiting .