Nocodazole arrest - yeast

[yeasty_boyz]

I (as well as everyone in the lab) have been having long arduous struggle with getting my S. cerevisiae cells to arrest in G2/M with Nocodazole. It's a fairly straightforward, std. protocol:
cells at 5E6, sigma Nocodazole in DMSO added at final concentrations of (since i'm titrating to find the right conc.):
10ug/ml, 12, 15, 20, 30 and 60. Obviously it's strain-dependent but I haven't gotten a single G2/M arrest with any of these concentrations. Incubation time is at least 1 generation. I also know Noc is tricky, but if anyone has any advice, please divulge...

sleepless in asynchrony

[Onyourcase]

Are your cells haploid?

[yeasty_boyz]

yup

[Onyourcase]

For cell synchronization experiments, nocodazole is usually used at a concentration of 40-100 ng/mL of culture medium for a duration of 12-18 hrs. Prolonged arrest of cells in mitosis due to nocodazole treatment typically results in cell death by apoptosis.

[yeasty_boyz]

Thanks onyourcase,
you were referring to yeast right? I know it differs for mammalian cells. My timecourse would not be longer than 6 hours. How long would it take for the cells to arrest? I suppose that depends on how quickly noc acts to depolymerize the microtubules. Once that is complete, the cells would arrest once they caught up to the next mitosis, correct?

[ololo]

It only takes 2 to 3 hours to arrest budding yeast in G2/M in nocodazole if you start from OD600=0.2 and grow it shaking at 200-250rpm. Final concentration in media is 10-15ug/ml (can be used from 100x stock in DMSO). You need to add certain volume of nocodazole in the beginning and then re-add 1/2 of this volume every hour. And it works perfectly. Btw, cells do not like to spend too much time in this arrest, so they usually start to escape after few hours.

[ematosperdo1]

For nocodazole arrest of yeast cells (S.cerevisiae) your stock solution concentration should be 1.5mg/ml and final concentration on culture 15ug/ml, that is 10 ul per ml of culture, so i.e. if you got a 5 ml culture you shoud add 10ul per ml, from a 1.5mg/ml stock solution. What I usually do is to grow cells up to an O.D. of 0.6 or around that, and dilute them to O.D. of 0.2 or 0.3 and leave the cells for at least an hour or two, and after that time adding nocodazole treatment; after 2 hours of nocodazole treatment I add (another) half the amount of the initial concentration,so if at point 0 you added 50ul Nocodazole for a 5 ml culture, 2 hours later you have to add 25ul and wait another hour, so in total cells must be at least 3 hours in nocodazole treatment. One way of checking it, is to see at cell morphology, they should be dumbells with only one nucleus (stained with DAPI), other way is, if you have a Tub1-GFP(tubulin) tagged strain, check for tubulin depolymerization and if you got a Net1-GFP(rDNA marker) you should be able to observe what is called the rDNA loop. In anycase titration is a good option and varies from strain to strain, and you should check the specific conditions for your strain and experiment, but otherwise 3 hours under nocodazole treatment as specified before and controlling your O.D. will do in order to arrest your cells. DMSO final concentration in culture,should not exceed 1%. If you still got problems with that, try to prepare a new stock of nocodazole (nocodazole must be kept in DMSO at -20ºC in aliquots), maybe the batch was old or in bad condition, who knows.
here ther is a link that will help you : http://mcb.berkeley.edu/labs/koshland/P ... yeast.html

hope all this will be helpful, good luck

[schmittm]

Unless you are married to nocodazole, hydroxyurea works much better for yeast and its cheap.

[ematosperdo1]

Unless you are married to nocodazole, hydroxyurea works much better for yeast and its cheap.

It depends on what kind of arrest you want (phase of the cell cycle), Hydroxyurea works for S-phase arrest only, alpha-factor for a G1, and nocodazole for a G2-M arrest., it is not a question of just arresting cells but when on the cell cycle.

[jpatterson]

the length of time required to arrest a population of cells varies between strain backgrounds and really works best in YPD. Are you using synthetic media?