I have been trying to inject C. elegans without DIC (our lab can’t afford it at the moment).
I could see the gonad quite well under bright field. However, every time I jab the needle in and press the inject button, I could see the DNA solution either flushes the gut or the pseudocoelomic cavity. This is very frustrating as I could see the gonad but could never see the DNA flows and inflate the gonad making a u-turn.
Does anyone has any suggestion or trick to inject under bright field. Many thanks in advance
It is very frustrating and difficult to try and inject the gonad using regular optics. I was able to do so when I first moved to my current lab only because I had learned how to do injections with a good Normarski system in my previous lab.
I have two suggestions:
Practice injections using food coloring on nice fat worms with lots of embryos. As you figure out where the gonad lies, you can move to younger worms and use your DNA instead of dye.
If there is a worm lab nearby, see if you can go and learn to do injections at their lab with their good optics. Once you are familiar with placing the electrode properly when you can see well, you should have more luck using sub-par optics.
I’ve never done any injections without DIC, but I guess it might be very difficult.
As you see well the gonads, your problem seems to be to finding the right focal plane to do the injections, in order to inject in the gonad and not above or below it. Maybe you can try once you see well the gonad to move down (ou up) your needle little by little until you find the right place.
Janet’s suggestions are good.
Injecting with DIC is tough enough, I’d hate to do it without!
But I have done it with lousy DIC, coming in as an experienced injector. Remember that with a side view, one gonad arm is up, the other down. If you can figure out which is which, let that inform your guesses.
Also, I always start a new injecting person on a control plasmid into N2 worms, just to keep it as easy as possible. Maybe good to start there after the food coloring .
It sounds like you are close to getting it to work. Without seeing what’s happening it’s hard to be sure why you are having trouble getting into the rachis. One or more things could be contributing to the problem: Could be that your worms are drying out too much on the pad. You could try a lower % agarose when making your pads. If they are still struggling a bit after your injection that is a good sign, but if they are stuck flat and completely immobile that is not so good. Could be that your needle is not sharp enough; with a blunt needle you can often get through the cuticle but then have trouble piercing the gonad. How are you breaking your needle tips? How much pressure do you need to use to get flow? Another thing that can help is using a low angle of approach with the needle; that way if you go too far in you wind up penetrating along the length of the rachis instead of punching through into the intestine. And don’t let anyone sell you Hoffman Modulation Contrast as a substitute for DIC…
I made the agarose pad by putting a drop of 0.20% ~ 0.25% molten agarose on a glass slide and let it dry overnight. The worms seemed behaved well. I tested it by putting worms on the pad under oil for 10 mins.
So far I only had 1 success with injecting WT worms with a roller plasmid. It was a lucky one where I could see the DNA solution made a U-turn inside the gonad. That worm did produce 1 progeny with a roller phenotype! I never saw the blue moon again.
I am now injecting an unc strain (the Mos single copy insertion method). So far so no good.
I used the needles from World Precision Instrument, 1.0 OD, 0.58 ID and a Sutter puller P-97 (finally got access to these things from a lab far far away, I used the pre-pulled needles from Eppendorf before that and they were so expensive). I tried a range of conditions and settled on the one which produced needles with a tip that is just open enough and doesn’t need breaking and a pressure over 700 hPa is required to see a small DNA droplet flowing out (sometimes, the DNA flows out like a fine mist rather than a droplet).
And yes, someone was trying to sell us Hoffman Modulation Contrast while obtaining quotes!
The pads sound good. Congratulations on your roller! unc-119 is not so fun to inject, but you should check out Christian’s new paper since you could potentially inject into wild type and use Neomycin selection (Nat Methods. 2012 Jan 30;9(2):117-8. doi: 10.1038/nmeth.1865. Improved Mos1-mediated transgenesis in C. elegans. Frøkjær-Jensen C, Davis MW, Ailion M, Jorgensen EM.)
We switched to 1.5 mm OD/1.1mm ID needles since everyone in the lab likes those better, but that is probably not so important. If you can get flow at 10 psi (700 hPa) with an unbroken needle tip it is probably pretty blunt. I think you would be better off starting with a finer needle tip, then breaking off the tip slightly by poking it against something (I like to make a scratch down the middle of the pad using the corner of a razor blade since this creates little ridges of agarose). You will often get a nice beveled edge that will penetrate well and give good flow with approx 8-15 psi.
And, as Janet said, be sure to use hermaphrodites that have been adult for a day or so (and continuously on food - not from a chunk) since they will give you a nice big rachis to shoot for.
0.2% agarose? That seems low. I do 2.0% agarose in water, molten, then drop onto cover slip and make a sandwich. After congealed, I pull off the top.
I usually use these weeks or months later, and I have little problem with dessicating worms unless I try to do many simultaneously (i.e. > 6). Often if I use them next day, they’ll still be too moist and I can have a little difficulty getting worms to stick.