I have two questions. Your help is greatly appreciated!
On average, what is the maximum amount of time that you store your RNAi plates and then still use them? I poured, seeded and induced mine just over a month ago and am wondering if they are still usable? I poured a lot of plates at once as I was planning to set up a bunch of experiments back to back, but have been struggling with mold issues and so have had to clean up the worms and repour stock plates and now its been over a month since the plates were poured and seeded. What do you think - can these plates still be used for experiments? I did a positive control on this batch at the beginning but the positive control plates are all used up and all of the other plates are seeded already with the clones to be tested. I will be repeating this set of experiments again at least one more time (I already did one replicate), but really feel terrible about throwing out all of the plates I poured because of all the reagents that would go to waste and the time that it took to pour them. If anyone has used RNAi plates beyond 5 weeks and they still worked, I would really like to know. I understand that efficacy may be different for different clones, but I will not be using this experiment to definitively prove anything, but as a second of three replicates. The first replicate’s plates were fresh and I will make sure the third replicates plates are fresh as well.
This brings me to question two. I and several others in the lab have been struggling with mold. It seems to be mainly present on RNAi plates with L4440 and stock LB + carb plates with the RNAi clones. We’ve gone through and bleached worms, cleaned and autoclaved storage boxes, and wiped down our benches with alcohol, but mold is still growing on plates! Does anybody have any other more extreme suggestions on how to get rid of mold in the lab?!
For 2. I had a similar issue, I tried getting new streaks of my RNAi’s and empty vector. It seemed to help. I kept getting yeast on my RNAi plates. Also, how quickly are your plates drying? I have noticed that contamination increases when my plates take longer to dry so I try to get them dried as quickly as possible.
For 1. I think it really has to do with IPTG. Carb can last a while at 4C. We use ours for a few months, but also add IPTG to plates after seeding.
hurryscurry - the plates are drying just fine, I don’t think that’s the problem. I think it is either the L4440 clone itself or one of the chemicals most likely. When you add IPTG on top, do you add at the usual 1M concentration and spread across the lawn and not the whole plate? We add IPTG to the media. If anybody else has any experience with the lifespan of RNAi plates with IPTG added together with the other chemicals, please let me know.
Snug - I really hope not and I don’t think so! I really don’t want to add more chemicals to the mix unless absolutely necessary, but will consider it if nothing else helps - what concentration of nystatin do you use?
Yeah, there is IPTG in the media, but we will also add it on top of the plate after the seeded food has dried to super-induce. We don’t spread it on the plate, because we’re assuming it should diffuse throughout the plate overtime.
IPTG, I have been told, is the limiting factor of plates. And we use ours for 2-3 months, but we haven’t optimized this yet so we don’t know how far out IPTG can induce, nor whether the super-induction is even necessary.
Thanks, one last thing. To add insult to injury, I discovered this in the lab today. This LB is just days old Is this a related mold problem or a separate issue? Bottle was closed tightly and stored at RT. The other recent issue has been a weird looking lawn on all of my RNAi L4440 stock plates - see pictures in link below. Does this look like yeast/mold? (the RNAi plates I mentioned in my original post had distinct filamentous white colonies, not lawn that looks like in the pictures below, so that was definitely mold, but I am not sure if this is a type of mold too and if so, if it is related?) https://docs.google.com/document/d/1vNZ4knigydKZk5e8v9eXBDddxU7l4wUiniJjV9uMTHg/edit?usp=sharing
If you’re having so many contamination issues, I suspect that probably most of your stocks are contaminated. Like hurryskurry recommends, re-streak everything out. If you’re still struggling you can always re-transform your RNAi clones.
About what organism is on your stuff: It’s probably yeast. If your NGM plates are fine and you’re still having problems after re-streaking, try re-making and filter sterilizing your antibiotic and IPTG stocks.
Good luck. At least you don’t have the contamination that turns agar into mush. Worst!!!
Is this a related mold problem or a separate issue? Bottle was closed tightly and stored at RT. The other recent issue has been a weird looking lawn on all of my RNAi L4440 stock plates - see pictures in link below. Does this look like yeast/mold? (the RNAi plates I mentioned in my original post had distinct filamentous white colonies, not lawn that looks like in the pictures below, so that was definitely mold, but I am not sure if this is a type of mold too and if so, if it is related?)