I use uncs for microinjection. Before the injection, I transfer the worm on dried 2% agarose pad, into a drop of mineral oil. Very easy to pick (head or tail) - but then,
uncs do not crawl off the pick. I end up killing them all :-. My question is, how do you get unc off your pick?
Thank you
Dominique
Try using an eyelash or eyebrow hair glued to a toothpick (we use nail polish as the glue) instead of a platinum wire pick. I’ve found it’s much easier to get worms onto an injection pad using these pliable, thin hairs over the wire. It’ll take some getting used to but once you get the knack I’m sure you’ll get better results. Good luck!
I usually inject in a unc-76(e911) background, and don’t usually have this problem.
If it helps, what I usually do is as follows:
I have an injection pad prepared, which I’ve moistened slightly by breathing on it (this is in the protocol I learned from; I never tested its importance) and on which I’ve painted a thin strip of halocarbon oil using a Pasteur pipet.
I have a seeded plate with many 1-day-old adults. I place drops of halocarbon oil around the perimeter outside of the lawn, pick about two dozen adults from the lawn (picking the worms up on the bottom of the pick, sticking to it because of the bacteria) and put them down outside of the lawn, removing as much of the bacteria (and eggs) that I transferred with them as I can remove without going to much trouble. I then flame my pick, pick up a glob of oil from one of the drops, and use the glob of oil to pick up about twenty of the worms that I just moved, mostly on top of the pick. I can then transfer them from the pick into my stripe of halocarbon oil on the injection pad without too much trouble, by passing the pick back and forth through the stripe of oil, essentially rinsing the worms off. Getting them to stick to the pad is more trouble, and I usually wind up discarding 10-20% of the worms I transferred rather than continue to waste time trying to get them to stick. I usually arrange the worms so they are all basically in parallel to each other (perhaps as many as three abreast, but never with their sides touching) so all of them will have their gonads either facing right or facing left and I can inject every worm in two passes without reorienting the stage for individual worms.
Microinjection still isn’t fun, but this approach works well for me with Unc worms.
Note: I edited this comment when it was pointed out that I’d referred to “mineral oil”, but had failed to remember that I’ve actually always used halocarbon oil.
Hi Dominique,
I mostly inject unc-119(ed3) worms. It helps to flood the worms with injection fluid (after injecting into both gonads) - the worms dehydrate less and are a lot easier to get off the pad again. I flood them with enough liquid that they come “unstuck” from the pad. Like Hillel explains above, I can line up about 10 worms that way and get them off again. I also pick the worms into a drop of M9 on the recovery NGM plate to speed rehydration. Finally, I use a pulled glass pipette instead of a platinum wire. If you pull the glass thin enough it’s pliable and does not damage the worms. A hair glued onto a pick works well, too.
Good luck.
I always add 30 ul M9 (with a little Triton) to the worm, let it float up, suck it up, and then transfer to a plate.
Thank you very much for your suggestions!
Dominique-Seydoux lab-