My protocol didn't work when using SD-trp culture. And nocodazole may not work.
I detect the phase of yeast cells by flow cytometry.
I'm looking for a new protocol for synchronization of the budding yeast.
(To G2 phase)
Hi there,
There are 2 obvious issues to address:
the protocol might be unadapted (try the following one:http://torreslab.biology.gatech.edu/wp-content/uploads/2013/02/Budding-Yeast-Cell-Cycle-Arrest-Procedures.htm)
and/or
the strain is naturally escaping from the nocodazole induced arrest due to its genetic background (checkpoint mutant).
you may try to adapt the protocol to your specific strain for efficient arrest:
http://tfiib.med.harvard.edu/wiki/index.php/Yeast_Cell_Cycle_Arrests
Hi Xi and Dominique,
nocodazole does not work in synthetic medium.
https://www.ncbi.nlm.nih.gov/pubmed/3050491
it works if you add peptone to the medium (1% the best concentration), but in this way you loose the -trp selection, since in peptone there are basically all the aminoacids.
hope this helps,
Paolo
Perhaps try hydroxyurea. It worked for us, or if you're using a MATa cell type you can use alpha factor.
http://fg.cns.utexas.edu/fg/protocol__cell_synchrony.html
Best, PAM
Dear Dominique and Paolo,
Thanks for your answer.
I'v changed my protocol to the following one: (http://torreslab.biology.gatech.edu/wp-content/uploads/2013/02/Budding-Yeast-Cell-Cycle-Arrest-Procedures.htm)
It works better.
And I'll try to add the peptone to SD then. It's really help me a lot.
Thanks again!
Regards, Xi
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