There is a huge difference between mating on plates and mating in
liquid culture. You can get 25% - 80% of input cells to mate on
plates within 8 hrs on plates (YPD, for example). Letting the same
number of cells settle to the bottom of a culture tube for 8 hrs in
liquid YPD, followed by Millipore filtration and plating on selective
medium will convince you ERRONEOUSLY that you are dealing with a
sterile strain (mating down more than 5 orders of magnitude). Note
that there is nothing wrong with mixing cells from two liquid
cultures, Millipore filtering them, placing the filter on a YPD plate,
recovering the cells into water, and then plating dilutions onto
selective plates. That is how quantitative mating assays are done.
But the mating must take place on solid medium.
In liquid culture, you will get some shmooing but little or no mating,
probably because a-factor is so sparingly soluble in water. Grown in
liquid culture, the a-factor becomes bound to the glassware. In fact,
you can purify it by dumping out the whole culture, washing the
glassware with water, dumping that, and then extracting the glassware
with methanol. An alpha will only find an a when it is right next to
it on a plate!
Charles Brenner, Ph.D. Fellow of the Leukemia Society of America
Rosenstiel Center, rm 610 phone (617) 736-4944
Brandeis University fax (617) 736-2405
Waltham, MA 02254-9110 brenner at auriga.rose.brandeis.edu