I am desparately in need of some help with c. elegans microinjection. I amnew to the worm field and have been learning to microinject for about 3 months now and have had absolutely no success whatsoever. I have tried asking other people in the lab to help out and have tried troubleshooting myselffrom sources on the net or from books on microinjection but nothing has helped. I seem to be doing ok injecting the worms in the right place from what I can tell, my problem is the recovery of the worms. I use M9 buffer for recovery and they initially seem to recover really well, thrashing about in the M9, but once the M9 is absorbed into the plate and the worm adheres to the bacterial lawn, the head is still moving around a lot, but the back end of the worm seems almost paralysed and after a day or so the worm dies.So far the main advice I have had seems to be that there are two main reasons for the worm not recovering: the cuticle is irreparably damaged
during injection or the worm has dried out too much. I have tried to increase my speed of injection, I only inject one worm at a time and having timed myself typically take between 2-3 minutes to inject. I have tried tons of different thicknesses of agarose pad but nothing helped (I use 2% agarose in water). As for the damage to the worm, sometimes I can see this is a problem as the worm explodes after injection, but most of the time they seem absolutely fine, I have tried hundreds of different sizes and lengths of needles, tried breaking the needle straight on and at an angle, turned the worm at an angle to the needle so that only the smallest amount of the needle enters the gonad, but NOTHING has worked. Has anyone ever noticed this kind of thing happening before, I could really use some advice.