Troubleshooting Biolistic Bombardment

Model Organism: Worms Methods & Reagents
1

Hi,

I have have recently been trying to perform biolistic bombardment on DP38 worms with the unc-119 plasmid (I’m currently only using the unc-119 plasmid because the gene gun that we’re using is handmade and inherited from another lab, so we’re trying to get the protocol down), but I have not been able to recover any transgenic lines.

I think that the problem way lie with the procedure for coating the gold with DNA, particularly in my spermidine. I have been using spermidine from Sigma that comes in a 0.1 M solution (here: http://www.sigmaaldrich.com/catalog/product/fluka/05292?lang=en&region=US), which says to store at 2-8 degrees. Every protocol that I’ve seen says that spermidine is unstable in solution and should be stored at -20 for short periods, or -80 for longer periods. I’ve been storing it at 2-8 degrees, is there a chance that my spermidine is bad, and that’s what is responsible for my lack of results? The reason that I suspect that my spermidine may not be good is because of the amount of remaining DNA in solution after the preparation of the gold particles. To prep the gold, I do the following:

  1. Combine 50 µL of DNA (about 200 ng/µL) with 100 µL well suspended gold beads and vortex
  2. Add 150 µL CaCl2 and vortex well
  3. Add 60 µL 0.1 M spermidine and vortex for 3-5 minutes
  4. Tap spin and remove the supernatant
  5. Add 300 µL 70% ethanol, vortex well, and tap spin.
  6. Discard the supernatant.
  7. Resuspend in 170 µL 100% ethanol.
  8. Vortex vigorously for 5-10 minutes, then vortex lightly until use.

After step 4, I checked the supernatant with the NanoDrop Lite and it contained about half of my DNA still in solution (instead of on the gold!). Is this normal, or is it indicative of a problem with the gold prepation?

For reference, the protocol that I’m following is from wormbook, here: http://www.wormbook.org/chapters/www_microbombard/microbombard.html

Thanks so much,

Michael

2

We have found spermidine to be unstable when diluted in water. We buy spermidine alone (such as Sigma S2626) and then make a fresh solution in water each time. The spermidine vial can be stored at 4C where the spermidine is frozen (it is a liquid at RT).

You might also want to consider letting the gold, DNA, CaCl2, and spermidine mix incubate for a little longer. We place it on ice for 30 minutes before performing the washing steps.

We have never checked for DNA-binding to the gold, but my impression was that it is a pretty inefficient process.

Good luck!