I want to do large scale RNAi. Has any done feeding RNAi in liquid culture? I was thinking I will grow up the feeding bacteria Overnight then spin it down and add some IPTG in the culture.
Any ideas on how to do this? I need a lot of worms for the experiment I am doing.
Well, a quick search suggests that RNAi feeding in liquid works. A similar protocol from a different group can be found in Wormbook.
It isn’t really clear from the search results whether anyone has optimized RNAi feeding in liquid for large volumes (if optimization should be necessary).
Hi, we’re wrapping up a full screen using liquid-culture based RNAi.
We only needed a small volume of worms per gene so yours would be a bit different but we got the best results with this:
Culture RNAi in 4 parts Amp stock to 96 parts LB (500 uL) amp stock = 25 mg/mL
store at 37 degrees overnight.
first thing in the morning, make a fresh solution of 1 mM IPTG in LB and add 1 mL to culture.
Allow bacteria to induce for 4 hrs at 37.
spin culture(s) at 3k rpm for 2 minutes and remove supernatant.
resuspend bacteria in s-basal. Add worms that have been suspended in solution of IPTG+Amp in s-basal.
Store in a damp environment (we used a cardboard box with wet paper towels and a partially opened top) shaking at 100-150 rpm.
Hope that helps, the hardest part we had was getting a good balance of bacteria concentration. We resuspended bacteria in s-basal to halt their reproduction. Worms will die from lack of oxygen if solution has too much bacteria.
If you have any questions, my email is gsliwoski@mclean.harvard.edu
–Greg
I’ve done it a few times in half liter cultures and it seems to work fine. The way I do it is that I grow the worms in liquid culture with normal HB101 (op50 should do as well). Two or three days before I want to harvest the worms I spin down the worms and try to wash away the bacteria that have not been eaten. Then I put the worms in a new culture in which I have added the RNAi bacteria (no amp or IPTG are included in this).
The RNAi bacteria I normally grow overnight in the presence of amp and the next morning I add IPTG, to a final concentration of 1 mM, let them grow for 4 extra hours, spin down and store at -80 till I use them.
For embryos at least this works…