Biolistic Bombardment Trouble


I have spent the past two months attempting biolistic bombardment with no success. I have performed 6 bombardments using unc-119 as a selector and 6 bombardments using Hygromycin B as a selector, but neither have yielded any transgenic lines. To ensure that I’m not mis-screening, I also co-bombarded wild type worms with Hygromycin B resistance and a pharyngeal gfp marker once. We use a thermo scientific miniprep kit for our DNA preps.

I have checked several things that may have been responsible for the lack of results, which are:

  • Performed a restriction digest using Nhe1 on both unc-119 and HygR, which yielded the correct results on a gel (so the plasmids should be okay)
    • Checked to ensure that the plasmid is getting bound to the gold beads (resuspended some of the gold beads in water after prepping it and assayed the water using a NanoDrop Lite, DNA was present)
    • Synchronized the worms to be L4/young adult and used a very large quantity of them (confluent layer covering the plate, at least 1 worm thick)
    • Mounted some of the worms after bombardment to see if the gold particles were visible in the worms (it was difficult to tell because of the fine granules in the worms, but it appeared that gold was present)
      It should also be noted that I’m using a homemade gene gun inherited from another lab. The lab the gun was originally from has reported the generation of successful transgenic lines using this gun. The gene gun uses a swinnex millipore filter which is loaded with DNA in the center. We fire with the gene gun at a height of 7 cm and 400 PSI using N2 gas. We use 20µL of the gold/DNA complex at a time, and then fire at each plate 7x, in a hexagon around the edge and one in the middle, using a total of ~10µg of DNA. I have also fired at a height of 4 cm and 10 cm, although it doesn’t seem to make a difference.

Beyond this, I’ve been following standard procedures for bombardment.

It seems to me that I’m missing something really big (maybe a real gene gun…), as I would think that even an incredibly low efficient procedure should have produced results after 12 bombardments. Has anyone found any factor that seems to make bombardments just not work at all? Is there anything that I’m obviously missing/should check that I haven’t? Does anyone have any general advice for making bombardments more successful (or successful at all)?

Thanks so much,


I may have figured it out. Today while looking at some mounted worms under the scope I started playing with the contrast settings, and I think what I thought was gold before was actually just granules in the worms. I’ll play with the height and PSI settings of the gun some more to see if I can change that.

Hi Michael,

We use a biolistic transformation system (albeit Helium-based and with the pha-1 system) that is probably similar to the one you are struggling with at the moment, so I hope a few ideas, suggestions, 'what we do’s etc might help you through the impasse.

  1. One basic observation is that you should calibrate your gun if you haven’t already. Using just gold particles(no DNA) in the PVP carrier, shoot at a filter paper from the distance you think is right and have a look at the spread and form of the gold. draw a crosshair on your base to mark the impact point for future reference.

  2. Worms should be young adults carrying a few eggs (<= 10 eggs inside).

  3. We shoot at a mound of worms concentrated in ~1cm (restrict the area by spreading a small circle of OP50) rather than a thin layer.

  4. Cool your plates so that the worms slow and stiffen.

  5. Check the gold doesn’t clump together.

  6. We use 8 bar Helium for transformations.

  7. Vacuum is normally -0.5bar.

  8. Our pulse is ~10ms per shot.

  9. (Assuming you are successful) move the worms by chunking to a fresh plate and carefully transfer them (they are suffering).

  10. You see about 50% dead worms if you really hit them.

  11. It’s easy to see if the worms have gold particles inside them (body, gonad etc.).

  12. DNA per shot 10µg+

  13. Gold 0.5mg+

  14. Don’t leave the worms on ice too long!

  15. Younger worms reduce the transformation efficiency.

Hope this is useful.



We also use a homemade gene gun and it works fine. We have successfully used the unc-119 rescue (on DP38 and HT1593) and hygromycin B (IR83, IR87 and IR88, Pci digested). I agree with what Steve said and just want to add that we have observed decreased efficiency when worms have wandered too long in the lab (max 6-8 weeks after thawing). We also use Qiagen’s midiprep kit (ref. 50912143) for DNA purification. Though we have not tryed the one you mention, we have tried others and got no transgenic at all.

I hope this helps. Good luck!

Thanks for the responses! Bearing that in mind, it seems that we’re definitely not getting any gold into the worms (I almost never see any dead worms after bombardment). We have been using nitrogen gas for bombardments, but I think that we may need to switch to Helium, as I’ve seen several resources that say that helium is necessary to penetrate thicker cells (and certainly necessary to penetrate the worm’s cuticle!). If we start getting gold into the worms and still don’t see results, then I’ll try switching kits to see if that makes a difference.

Echoing what others have said, in my very limited and long-ago experience with the Helios gene gun, what I found to work was to start with a large pile of worms (unc-119 is very good at forming large piles) and hit them with a blast of gold particles at high enough pressure to shred the top layer of worms and pepper the lower layers with microparticles. Shots at still higher pressure settings blew the worms off the plate; if you can find a pressure setting at which that happens and then back off, you might be in good shape.