We have been injecting 1 day adult worms for last two months and have been struggling to obtain enough F1s though we have injected over 70 P0s sometimes over 100 P0s. Our injection efficiency seems to be pretty low and we have tried RF4 (25 ng/ul) ans CC-dsRED (25 ng/ul). Our plasmids seems be very clean and have obtained F1 and F2 with low numbers.
Our concentration is as follow
Total of 200 ng/ul
pBluescript 174 ng/ul
Co-injectiontion 25 ng/ul
Sample 1 ng/ul
I would say that your concentration is higher than normal. This should not cause you to get F1s any less often (if anything, the reverse), but it’s high enough that I’d expect some trouble doing the injections, I’d expect your needles to clog frequently. If that’s not happening, I wonder about your injection technique - maybe you have needles that are too blunt / too fast a flow rate, and are having trouble because your injections aren’t going well? Do your injected animals often fail to recover well? Do they give you many progeny?
20-30 F1s is not very good. Ideally the worm should be healthy and fertile for a couple of days after the injection. You may be getting progeny that are almost entirely the embryos and maybe differentiated, cellularized oocytes that already existed when you did the injection.
I haven’t looked around for good injection videos (there’s a JoVE article, but it seems to skip the actual step of flooding the gonad; it may be hard to capture on camera). You want a needle that enters the worm fairly easily, with a light tap, and flow that isn’t too rapid but also isn’t so slow it will immediately clog. This is hard to describe, and really is a matter of trial and error. Then, when you inject, you want to see the syncytial gonad become turbid and maybe darker/different in appearance, flowing out from the injection site fairly rapidly but not in a flash, and stop as it approaches the turn.
There is a strong community bias in favor of Qiagen (Qiaprep, QiaQuick, or Qiagen miniprep) but I don’t know if there has even been a careful head-to-head comparison.
Omission of RNaseA in the P1 buffer and ethanol precipitation of the plasmids boosted transgenesis. He also reported that the cheaper Fermentas GeneJet kit worked comparably to the Qiagen kit. He reported that there was little to no RNA in the miniprep (at least by agarose electrophoresis and EtBR staining).
For genome editing, Dan Dickinson recommends the Invitrogen PureLink HQ miniprep kit and including the optional wash with 4 M GDN-HCl + 40% isopropanol. I’d either try that kit (works well for us) or the Maduro “omit RNaseA and do an ethanol precipitation” approach.
Just leaving out the RNase from the preparation kit, in my hands, made all the difference between almost no F1s and a lot of them. It is worth a try if nothing is working.
Also you could get some ‘working DNA’ from another worm lab near you and see if you can get F1s with their prep.
I would definitely take a step back and focus on just getting a good number of F1s with either pRF4 (rol-6) into N2 or unc-119(+) into unc-119 mutants. Both of these plasmids seem to be well-tolerated and work at low to high copy number. Once you start adding in other plasmids it gets tougher to pin down a problem on injection method vs. transgene toxicity.
If you are new at injections, I recommend practicing on N2 with a rol-6 marker rather than unc-119 which is harder to recognize as damaged. A good standard injection is when N2 crawls normally on the recovery plate and gives hundreds of progeny (if you injected young adult). Finally, could over expression of this gene be toxic? I would try a lower concentration.
Good luck!
In our lab we use the Quantum miniprep kit from Biorad when we miniprep our DNA. We also filter the made up injection mix before injecting into our worms.
We use the Ultrafree-MC Centrifugal Filter Devices from Millipore. We use to not filter and saw that injections worked about 80% of the time. Then we started filtering and have had no problems.
I strongly endorse the use of a filter. It was only a few years ago when somebody recommended to me. My clogging problems plummeted. $1-2 per column. But the improvement in efficiency made that SO much worth the small cost and time!