I’m using the plasmid from the library which contain the Bli-2 gene, and I can not see the phenotype reported at the wormbase
(phenotype observed with the interference of Bli-2). The clone I am using is JA:F56C11.1.
I sequenced the clone, and it is okay, and my positive control is dpy-11, which works very good.
The protocol that I use is:
inoculate RNAi clone to LB Amp plate
pick colony and inoculate LB Amp broth
seed 5 cm plates ( NMG with 1 mM IPTG and 100 ug/mL) with RNAi bacteria and 1 day at 37°C.
-bleach the worms and add the synced L1 or unsynced eggs straight onto seeded RNAi plates.
Have someone worked with this clone and have seen the blistered worms (one of the phenotypes reported)?
I can’t speak to why the clone isn’t giving you a blistered phenotype, although you may want to try an RNAi-sensitized strain
like the one used in the Simmer F et al 2003 PLoS Biology paper (WBPaper00006395). The strain is:
I did notice that the clone that you report using (JA:F56C11.1) is reported to target the bli-3 gene, not bli-2. The appropriate
clone to target bli-2 would be JA:F59E12.12 (Source BioScience: II-4F24).
I had a similar thought to Chris’s, but the phenotype section of the bli-2 gene page reports a Blistered phenotype for RNAi treatment citing not only the Simmer paper (rrf-3 RNAi-hypersensitive background) but also the Kamath paper (wild-type background). Although, the two may have differed in terms of penetrance or expressivity. It may be relevant that you’re RNA’ing from L1 on, but as I recall (ie without bothering to check) the RNAi screens started at least a generation earlier.
I assume you’re familiar with the bli-2 mutant phenotype? If I recall correctly, it’s adult-specific …
I noticed you did not include tetracycline for your bacterial culture. The RNAi bacteria include a transposon insertion in an RNase gene, thus stabilizing the dsRNA for RNAi. The transposon has tetracycline as a selectable marker. Although unlikely, it is possible that by omitting tetracycline you have lost this key insertion and that your bacteria are not competent for efficient RNAi. Try adding tetracycline to your cultures, at least to the bacterial plates.
Many thanks for all your answers. I will have in mind all the suggestions.
Chris; it´s okay what you said, the target gene is bli-3, I made a mistake when I was writing the post, but I do know it´s bli-3 and not bli-2.
HillelSchwartz; As you said the blistered phenotype it´s adult-specific. I am starting with eggs but I follow up to the F5 generation.
Sven; I didn´t include tetracycline, I will do it. It´s only added to the bacterial plate (not the broth), isn´t it?
What do you thinnk if I induce with IPTG in the bacterial broth culture?
Should I try increase the IPTG concentration?
You are right - tetracycline is often only included in the bacterial plate. However, some groups report that it is best to use tetracycline in the broth as well. Try both approaches? My lab uses tetracycline for both steps of bacterial culture and even on the worm plates, which works well for us. Regarding IPTG addition, the 1mM IPTG induction on worm plates for 24-48 h at RT (in the dark) seems to work pretty well. Good luck!
I’d also try a positive control with something else. pop-1 has been popular in my group: effective RNAi kills 100% of embryos. Plate the parents at L4 and transfer after 24 hours, and you should get a plate full of dead embryos. Also, we use oil to transfer the worms, to minimize the OP50 that gets picked over to the RNAi plate.
try incubating the seeded plates overnight at RT instead of at 37 degrees.
Is it possible that the Bli genes you are targeting has maternal effect transcripts that are somehow not targeted by RNAi? Do you see the phenotype in the next generation?
When I do RNAi screens, I need to feed L3-L4 and then look at phenotypes of the next generation as it tends to show more phenotype than if I feed L1 and assess phenotype in the same generation. It is gene-dependent, though.