Does any one know how long it takes for Thymidine analogues to be uptaken from media until being incorporated into newly synthesized DNA? I work with S. pombe and I would like to do a double pulse labeling of DNA. But its tricky to know how long in advance add the second analogue. Is it taken into cell instantly or does it take some time?
Thanks!
Hi Parham,
If I understand your question correctly, you are asking what is the shortest time between addition of the analogue to medium and incorporation into DNA assuming, of course, DNA synthesis is going on. I have no experience with yeast, but in mammalian cells it takes very few minutes at the most. You can pulse-label the cells for 15 minutes (from addition to medium to fixation for staining) and still see a very clear signal. Also, still in mammalian cells, you can use two labels one after another with a simple wash in between and there is no overlap and no gap between the two labels (used for DNA combing). Thus, in mammalian cells, uptake and washout are very fast. I'd assume it's essentially the same in yeast.
I hope this helps
Hi Parham,
It takes some time for the incorporation, because depends on the cell proliferation rate.
I think you should try a curve adding the second pulse between 3 and 24h.
Good luck!
Consider the dividing speed of your yeast. At least one cycle needs to incorporate the analogue (because of the synthesis of new DNA).
Hi Parham,
It depends on doubling time. It is also important dividing cells to be synchronized. I work with adherent tumor cell lines and harvesting in logarithmic phase of growth by trypsinization and seeding into new flask synchronizes most of the cells. I did experiments only with one analogue (tritium thymidine) and the incubation time was 24 h (or at least overnight). Medium is also important (type and changing).
Good luck and please tell us when you succeed and how. This information is precious.
Best regards
Hi Parham,
If I understand your question correctly, you are asking what is the shortest time between addition of the analogue to medium and incorporation into DNA assuming, of course, DNA synthesis is going on. I have no experience with yeast, but in mammalian cells it takes very few minutes at the most. You can pulse-label the cells for 15 minutes (from addition to medium to fixation for staining) and still see a very clear signal. Also, still in mammalian cells, you can use two labels one after another with a simple wash in between and there is no overlap and no gap between the two labels (used for DNA combing). Thus, in mammalian cells, uptake and washout are very fast. I'd assume it's essentially the same in yeast.
I hope this helps
Myself have not done this labeling, though also worked with pombe quite a bit. Looking at the literature, this link should help you:
http://www.ncbi.nlm.nih.gov/pubmed/25916707
Thanks everyone for your answers. Marco you are absolutely right. That's what I am intending to do and my strains have hENT1 and hsv-tk , so they should incorporate analogues almost at same rate as mammalian cells. What you pointed out is not very clearly mentioned in protocols, that how fast is the uptake and wash out. Because I am trying to do the pulse labeling too. So what you do is that you add the analogues in middle of S-phase not prior to it? That's the only difficulty with pombe since the s-phase is much shorter than mammalian.
Thanks a lot !
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