I am doing lifespan assays for ~35 genes simultaneously in C. elegans on FUDR (inhibiting reproduction) plates to avoid re-picking the worms. I tried it once and got positive results, but a problem is that the worms occasionally burrow into the NGM gel, sometimes due to my accidental damaging of the gel, sometimes for no apparent reasons. Now I am trying to do the experiment again and thinking about increasing the agar concentration to strengthen the gel to reduce the chance of worms getting inside.
However, senior members in the lab are not comfortable with the change because the agar concentration of 1.7% is the standard and everyone is doing it that way. The question is, how is the 1.7% agar established? What is the reason for that concentration? Are worms still comfortable in higher concentration, such as 2.5%, of agar? Does that change impact lifespan?
For my lifespan assay, I am not looking forward to comparing with other labs. I am simply comparing two groups of genes (evolutionarily selected by long vs. short lifespans) to see their different impact on lifespans after RNAi knockdown. For me, it seems that the experiment is well controlled and whether the agar concentration is standard is almost irrelevant as long as the worms are not stressed. Given the huge amount of work each day and length of time worms have to be kept on the same plate, I'd rather go for a smooth process without many accidents than sticking to the standard. I also have a very limited period of contract (employment, only a few months), so I hope I do not have to test it out first.
So, if someone has the experience of a different concentration of agar, knows how that impact lifespan assays, or can point to some materials that explain the standard, that will be greatly appreciated.
Thanks!
Dear Jiang-Nan Yang,
I have tried upto 2 % of agar concentration in NGM plates and I couldn't see any drastic changes in their locomotion or behavior. I don't think it may create any stress over them.
In my lab, this 2.5% has made as standard concentration for NGM. It doesn't make any stress so far to the animals. But I'm not clear about the temperature that you weren't mentioned above anywhere else. So it's important to maintain the environmental temperature for the animals since they can't sustain up to high temp. Just also check with the proper temp for that particular Mutants from the Research articles
Venkatakrishna LM Thanks for the answer. Is a 2.5% gel more difficult to make than 1.7% gel? Does the higher concentration result in more easily formed air bubbles that eventually would break the gel integrity and let worms burrow inside anyway? Is there any other technical issues I have to consider?
For temperature, we use 15C.
Jiang-Nan Yang The air bubbles might form during transferring the media to the petri dish but those do nothing for the animals because you're going to do the experiment for may be a week! So you need to be just careful while your preparations. For better, pour 70% of NGM to the capacity of plate. I would again suggest you that try with 2.5% in your preparations.
Now I can give some feedback. Following to the publication here :Article A Simple Method for High Throughput Chemical Screening in Ca...
I used a gel concentration of 2.3%. It was more difficult to make during melting the gels but bubbles are not a concern. Not so many formed. What is very interesting is that worms do not burrow inside the gel even if you damage it, making it very easy to handle worms. I saw no negative effects on worms.
However, be aware of the edges and never add liquid (culture or water) to the junction of the gel and the plate wall. Otherwise, the gel would detach and worms will crawl inside, especially when the gel is drying up.
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