Hi everybody,
I'd like to use nocodazole in a selective synthetic medium (I mean: containing just a selection of aminoacids), to trigger an inducible promoter in saccharomyces cerevisiae.
nocodazole is not working in the 'classic' synthetic medium, that is the one made with water + aminoacids + glucose + YNBs.
if I add peptone to it, noco works: but since peptone also contains aminoacids, no induction is possible: it thus cannot be my solution.
is anybody using nocodazole with synthetic medium?
how is the medium prepared?
do you keep some particular pH or similar stuff?
thanks a lot in advance,
Paolo
Dear Paolo,
I'm not familiar with nocodazole but I used a lot another azole agent (ketoconazole) and have found that, in synthetic medium, the pH of the medium AND the intracellualr pH of yeast cells become acidic. This have some impact on some processes looked at.
One way we have to overcome that is to buffer the culture medium. This is what I suggest to you. Just buffer your culture medium, with, say, phosphate buffer added and see what happens. I'm sorry to give you this solution which is empiric.
Best regards,
Philippe
dear Philippe,
thanks a lot for your answer: the same suggestion came from a colleague in the lab, so I tried: I set pH to 7 (as our YEPD) using NaOH at the beginning of the experiment, and kept track of pH change along the exp.
pH changed a bit (and got more acidic, as you said, reaching around 6.5) both in YEPD and in my Synthetic Buffered Medium. having the same pH as YEPD did not help nocodazole to work: immunofluoresce angainst tubuline showed wonderful microtubules!
do you know if any specific transport pathway is involved? or some chemical degradation is needed for 'azoles' to work? I read something about this last idea, I can't remember where..
thanks again!
Paolo
monosodium glutamate is needed for antibiotics to work in Synthetic Medium:
http://science.sciencemag.org/content/sci/suppl/2004/02/05/303.5659.808.DC1/Tong.SOM.pdf
I don't know the chemical details, but it might be the same for other compounds?
if someone has experience with that please tell..I'll try anyway
for those still interested I found this paper:
https://www.ncbi.nlm.nih.gov/pubmed/3050491
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