I’m working out an RNAi protocol using a synchronized culture of worm embryos for an undergraduate molecular biology laboratory course. It seems that nothing will go right with this project. I’m using the L4440 plasmid as the control and using the plasmid from the library contain the Bli-2 gene.
The RNAi wouldn’t work with the N2 strain so I tried using rrf-3 and eri-1 mutants but the RNAi still fails. I’ve sequenced the plasmid and the gene is present, I’m going to be doing some more sequencing reaction because there only seems to be a 95% alignment to reference.
I’ve switched up the ITPG source when making the media to see if that was the problem.
Are there other experiments I could do to narrow down the point of failure? I didn’t think a few point mutation in the bli-2 gene would affect the experiment but now I’m wondering.
One suggestion would be to get a few other good control RNAi strains to see if you have a general issue with your RNAi or if the problem is specific to bli-2 RNAi. I’d recommend some of the following:
-par-2 (should cause Emb phenotype)
-unc-22 (twitching Unc phenotype)
-GFP RNAi (if you have access to a fluorescent scope and a strain with a bright GFP transgene)
-let-767 (lethal)
-dpy-9 (dumpy bodies)
-cdc-25.1 (Sterile, germlineless animals)
These have all worked well in my hands and should give you an indication as to where your problem lays. Other ideas are to grow the animals multiple generations on RNAi plates. How do you grow
your bacteria for RNAi? ie. do you induce in the culture that you seed, or induce on the plate? How much IPTG are you using? Are you including tetracycline in your plates? These are all variables
that could effect your efficacy.
Try using gon-1 as a positive control. The worms are not able to develop gonad tissue, they are sterile, sickly and have a large protruding vulva phenotype.
The protocol that I use is something like this:
Day 0: inoculate RNAi clone to LB Amp plate
Day 1: pick colony and inoculate LB Amp broth
Day 2: Seed 5 cm plates (1 mM IPTG important to add IPTG after media has cooled a bit, but not solid) with RNAi bacteria
Day 3: egg prep worms and place ~200 eggs to seeded RNAi plate
I usually am looking at late L4s so that’s like 44-48h at 23C.
I hope this helps. If gon-1 doesn’t work for you, you’ve got some problems elsewhere.
Always use a positive control for RNAi! I have found that under optimal conditions, pop-1-directed RNAi confers 100% lethality (mostly embryonic but the occasional L1). As conditions become suboptimal (say, old plates), the lethality drops. So, a nice sensitive positive control for good RNAi conditions.
To add to what others have said, I’d be careful to wait until agar has cooled to 50-60C before adding IPTG or Ampicillin. A good rule of thumb with plate additives.
And as JordanWard says, one gets best RNAi by picking from relatively fresh bacterial streaks (on Amp+Tet plates), growing overnights with Amp but not Tet, not growing overnights too long (<=16 hours), and spotting on plates with IPTG and Amp (or Carb) but not Tet. I haven’t played with conditions a lot, but anecdotes say this protocol works best.
Also, I plate late L4s to plates without food, let the worms swim out of food smears, then pick using heavy mineral oil to minimize foreign bacteria. I grow overnight and then transfer, since some of the first progeny on pop-1(RNAi) will survive. I generally get 100% lethality on the transferred plate.
we always transform our L4440 plasmid before starting a feeding experiment. From the transformation, we start the bacterial liquid culture, induce at OD-= 1 to 2. with 1mM IPTG.
We then store the feeding plates not more than a week at 4°C. We noticed that for many genes, RNAi is less efficient when bacteria come from glycerol stock or platsecond streaked plates.
where does your feeding vector come from ? some vectors are better than others, although the sequence is ok. You may consider creating a new vector.
choose another piece of the gene, or just reclone it. It can help.