Dear all, I'm able to synchronize yeast haploid (a type) with alpha factor it is working fine.but i don't know how to synchronize yeast alpha type as well as diploid cells. I'm looking for some information about synchronizing of yeast diploid cells as well as haploid alpha cells with what sort of things.
Hi there,
Hydroxyurea blocks cells in S phase (use it at 0.1M for 2h). Nocodazole blocks cells in G2/M phase (15µg/mL for a couple of hours). For these two drugs the arrest phenotype is large budded cells (dumbbell) so you can check the block efficiency by bud index. Sonicate the sample before bud index.
Thank you Liger for your suggestion, we used Nocodazole also but it is not worked in our case. As you said above that how can we check the block efficiency by bud index ?
Hi again,
bud index is obtained by counting budded cells in a sample under the microscope. You need to count around one hundred cells and calculate the proportion of budded cells. If the block is efficient the index gets very close to 100%. But if you expose the cells for too long they tend to escape the block. And as I said earlier it is important to sonicate the sample in order to separate cells before counting.
Thank you Liger , actually i need to check the synchronizing cells in FACS analysis , if you don't mind can you send me any protocol by which u people r synchronizing by using NOCODAZOLE n Hydroxxy urea.
hi again,
Have a look at this:
http://fg.cns.utexas.edu/fg/protocol__cell_synchrony.html
Hope it helps.
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