I’m trying to make RNAi work on my bench, so far no positive phenotype observed… I’m using N2 and unc-22, dpy-6 etc. so my guess is that the antibiotics or IPTG went wrong.
And I just read some papers saying that IPTG is light-sensitive and should be kept in dark. How does this work when they’re getting seeded? When I seeded them I just left them on bench top overnight, and collect them next day.
How do you normally handle IPTG plates in your lab?
I do not know how much light is needed to significantly degrade the IPTG. But, just to be careful, I do cover the plates on the counter loosely with a couple of layers of aluminum foil. And be sure to use the IPTG+amp plates shortly after you make them. You can find suggestions on the best way to make your plates by searching this forum.
I sincerely doubt that light is causing your RNAi problems.
I would try some additional controls, including ama-1, and go back to some of the original papers to get your induction conditions straightened out. The Timmons et al. (2001) paper in Gene has a particularly thorough test of conditions that include a liquid culture induction by IPTG followed by seeding to plates (that I believe also have IPTG).
The tetracyclin in the plate is what is light sensitive. IPTG has a relavitely short half-life, so you should keep plates at 4 deg until you seed them, and then seed what you need and use them soon after. Also, you can add IPTG to the cultures themselves as you grow them to get a jump start on the induction. Good luck!
I’m now preparing a new batch of plates and bacteria cultures based on your suggestions - keeping plates in 4 degrees until seeding, covering plates with aluminum foils, adding in IPTG to overnight cultures before seeding (found the protocol here! http://forums.wormbase.org/index.php?topic=1314)… Hopefully this will work!
Actually I had a few plates with positive phenotype, but not all plates seeded with the same bacteria responded. I guess - for whatever reasons - there’s some variation between my plates that killed the pilot. I found another protocol of IPTG plates here http://130.15.90.245/rnai_plates.htm that they directly apply IPTG & amp on already-made plates, which perhaps reduces variation between plates. Is there anyone following this protocol?
