Yeast Liquid culture - 2 things (Apr/27/2005 )

1. size of vessel and volume within

someone told me it is important to leave 10 times as much air in the vessel as you have liquid
i.e a 10ml cultre should be in a 100ml flask
is that true?


2. yeast under selection are meant to "dump" unwanted plamids if this selection pressure is not mainatained, I screened a library ina yeast with another plasmid, the other plasmid contains a reporter gene, I of course want to plasmid rescue only the library plasmid, so instead of growing in -u -t media, i only gre the yeast in - U only media to force the yeast to "dump" the -T plasmid, however after 3 days in -U media, almost all the recovered plasmid was not the library plasmid pFL61 but the plasmid (pYES3CT) containing - T selection with the reporter gene

any comments?

1. The reason why you keep the size of the liquid volume small in comparison to the air volume is to maximise the surface area for gas exchange. As the flask rotates the liquid media coats the surface of the flask, gas is exchanged and then the coating is washed back into the bulk of the liquid, which coats the surface etc, etc, etc. So>>yes it is true that this 10 to 1 rule should be approximately followed.

2.The idea that yeast cells "dump" their plasmid is true but rare. The yeast cells are just as likely to dump your library plasmid as the other one. If the library plasmid is at lower copy number and is more expensive to the cell to maintain (larger, codes for deleterious proteins etc) then this method is more likely to select cells that maintain the second plasmid only. Yeast dump plasmids at random and if this lack of plasmid gives a growth advantage then that clone will come to dominate the culture. I'm sorry but I don't not think your strategy will work. You may eventually select out a clone with only a "library" plasmid but this will be a product of selecting for growth advantage and not necessarliy what you originally screened for.