I’m considering using FUDR in some experiments on age-dependent phenotypes, but I have no experience with FUDR protocols and I was hoping for some advice on the matter.
I’ll be rearing my worms on NGM plates; my question is about how best to apply the FUDR to the plates.
I’ve come across several protocols/papers that describe adding FUDR to melted agar prior to pouring/drying. However, a few papers appear to describe adding the FUDR to the already poured/dried agar plates – plates with OP50 and worms already on them. This latter approach would be much more convenient for my present experiment. Unfortunately, I’ve not found any papers/protocols that either describe this method in detail or compare the efficacy of the two approaches.
Does any one have any experience with this approach? If so, I’d be happy to read about how it was done, how well it worked, etc.
Our lab has never tested it, but I would stick with the tried and true and adding it to molten agar at around 55C, when you add the salt solutions, as what most (apparently most) labs do.
edit: We have successfully performed experiments using titrations of FUdR by adding it to the media at 55C, so I doubt that adding it in at this temperature is negatively affecting the compound. We do thaw the tubes at RT though.
Wait a sec…the worms are already on them? Are you sure? Do you have the refs? If so, how do the authors ensure that they’re spreading the compound equally throughout the agar surface (can’t really hockey stick it around unless you wanted to harass the worms, haha)? I guess it would diffuse through the agar, but I’d be a little skeptical still about the handling…
Adding it to the molten agar works well because you know exactly what the concentration of the compound is in the media. Also, how fresh will these plates be? I find that old plates shrink and you’d lose volume (and thus be harder to control for how accurate your concentration of FUdR would be), and sometimes even plates poured with a slightly mis-calibrated plate pouring machine can give you off results…
The practice of adding FUdR ‘to the media’ with worms already ‘seeded’ is more generally done for worms grown in liquid culture. I have seen a couple studies where people have added FUdR to solid NGM but usually in small volumes and only using a multi-well plate format (6 wells+++). I agree it may be a bit harder to ensure that the FUdR is going everywhere on individual NGM plates if you don’t apply a large volume.
You could also argue that the worms ‘see’ a pretty high concentration of FUdR as soon as you add it to the agar and before it soaks in and diffuses through the plate. This could potentially be…not good.
Thanks for the thoughts, everyone. Much appreciated.
Here’s one (see the section “Postdevelopmental RNAi lifespan screen”):
Curran SP, Ruvkun G (2007) Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability. PLoS Genet 3(4): e56. doi:10.1371/journal.pgen.0030056
As for how they ensure that the FUDR is equally distributed, I cannot say. But, as JMacIntyre suggested in his post, they’re using a multiwell plate, so maybe they’re simply assuming that local concentration can’t vary much given the smaller well volume.
I was thinking more like Sylvia’s paper (http://www.nature.com/ng/journal/v33/n1/full/ng1056.html). From what I understand, they did their screen using FUdR on 24-well plates, adding it to adults.
Once they got their hits, they performed more ‘clean’ lifespan experiments on regular single NGM plates and then transferred adults to NGM+FUdR plates. I think this more ‘clean’ way is the way to go.