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alpha factor synchronization

Hieter Lab Hieter at jhuigf.med.jhu.edu
Sat Oct 8 14:23:14 EST 1994


In article <pablo-061094155931 at 128.42.239.32>, pablo at bioc.rice.edu (Paul
Campbell) wrote:

> We have been trying to use alpha factor to synchronize S.cerevisiae, but
> the results have not been satisfactory.  We are using bar1 strains and
> 1ug/L alpha factor in mid-log phase cells for 2.5 hours at 24C.  The alpha
> factor is filtered out, and the cells are then washed in 2 volumes of YPD
> and resuspended in the orginial volume of YPD and grown up at 24C.  From
> the bud index taken at
> various times after release, the cells do not appear to be as well
> synchronized as they should be.
> 
> Any suggestions/how do you do your alpha factor synchronizations?
> Thanks!

We would suggest the following modifications:

1.  Arrest them for a longer period of time at 24¡C with alpha factor. 
Perhaps 3 to 3.5 hrs.

2.  Wash the cells much more extensively (at least 4 times) before
releasing them into YPD, and use cold water rather than YPD.

What is the problem anyway?  Do you have a large population of budded cells
even before releasing, or a large population that doesn't release?  Please
be more specific with your problem....

J. Kroll And S. Tugendreich



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