Creating Integrated Lines using Microparticle Bombardment
(adapted from Austin lab protocol and Biorad manual)
Note: preparing the gold beads for bombardment involves a lot of vortexing; a turbomixer attachment for your vortexer is strongly suggested (available from Fisher). Both gold and tungsten beads work, but a rigorous comparison has not yet been performed. Store dry tungsten and gold microcarriers in a dry, non-oxidizing environment to minimize agglomeration.
Preparing the beads
- Weigh out 30 mg of microparticles into a 1.5 ml microfuge tube.
- Add 1 ml of 70% ethanol (v/v).
- Vortex vigorously for 3–5 minutes.
- Allow the particles to soak in 70% ethanol for 15 minutes.
- Pellet the microparticles by spinning for 5 seconds in a microfuge.
- Remove and discard the supernatant.
- Repeat the following wash steps three times:
- Add 1 ml of sterile water.
- Vortex vigorously for 1 minute.
- Allow the particles to settle for 1 minute.
- Pellet the microparticles by briefly spinning in a microfuge.
- Remove the liquid and discard.
- After the third wash, add 500 µl sterile 50% glycerol to bring the microparticle concentration
to 60 mg/ml (assume no loss during preparation).
The microparticles can be stored at room temperature for up to two weeks. Tungsten aliquots should be stored at -20°C to prevent oxidation. Gold aliquots can be stored at 4°C or room temperature.
Putting DNA onto beads
All amounts listed in this section are per hepta-bombardment. I adapted this from the Biorad protocol keeping the CaCl2 and spermidine concentrations the same.
- Vortex prepared beads in 50% glycerol for at least 5 minutes.
- Put 60 µl beads into siliconized eppendorf tube. Do this quickly, before beads settle.
- Start vortexing beads and make following additions in order listed (vortex 1 minute between each addition, stopping the vortexing as briefly as possible):
- 10 µg DNA in 50 µl
- 100 µl 2.5M CaCl2
- 40 µl 0.1M spermidine (store frozen at -20°)
- After additions are complete continue vortexing at least 3 minutes.
- Allow beads to settle for 1 minute.
- Spin briefly (3-5 seconds) in microfuge; remove supernatant.
- Resuspend pellet in 300 µl 70% EtOH. Note: if there is a ring of beads on the tube wall scrape it off with pipet tip.
- Spin briefly; remove supernatant.
- Resuspend in 500 µl 100% EtOH (use water free EtOH).
- Spin briefly; remove supernatant.
- Resuspend in 170 µl 100% EtOH (use water free EtOH).
- Vortex at least 3 minutes. Continue resuspending beads by vortexing and pipeting up and down until all large clumps of beads are broken up. Note: resuspending the beads is difficult because they are crosslinked by DNA. Vortexing by hand instead of turbomixer may help with resuspension of beads. To test resuspension, put 1-2 µl on slide and view with compound microscope. You should see single beads and small clusters (1-10 beads); if you see mostly larger clusters, continue resuspending.
Loading the microcarriers
The microcarriers are very static and will stick to anything. I like to put a bunch of them in a dish of isopropanol, then pick them out with a pair of tweezers and put them on a kimwipe to dry. When they are dry, I put 7 in the lid of a petri dish. Then I add 20 µl beads per microcarrier and let them dry.
Preparing the worms
You will need ~100,000 worms per bombardment. I find it easiest to grow the worms up in liquid culture. I bleach worms, and put up to ~500,000 eggs in 100 ml S-medium in a beveled flask, on a shaker at 20°C. The next day, I add OP50, nystatin and penicillin/streptomycin. When the worms are young adults, the are ready for bombarding.
Bombarding the worms
These are the setting used by the Praitis lab for the Bio-Rad PDS-1000/He:
- Gap distance: 1/4" (decreasing gap distance increases bead velocity)
- Rupture disc: 1350 psi (higher pressure rupture discs increase bead velocity)
- Vacuum: 28 inches Hg (the vacuum serves to increase the pressure differential)
- Target shelf: second shelf from bottom (the machine works like a shotgun: the further the distance the beads travel, the more they spread out).
Post-bombardment care of worms
- Allow worms to recover for ~ 1 hour.
- Wash worms off of each bombardment plate with M9 buffer and put them on 5 or more 100 mm plates. Save original bombardment plates, as these will occasionally also yield transformants.
- You can look at worms under the microscope; beads are visible at 100X.
- Screen for transformed animals after 10-14 days at 20°. You can check earlier but it takes a while for the untransformed unc-119 worms to starve and die. In addition, early transformants are more likely to be transiently transformed. By 14 days post-bombardment, it should be easy to pick out non-Unc transformed worms.
- Clone out 10 -15 animals from each plate that contains transformed animals and look for clones that do not segregate Uncs (or that segregate 1/4 Uncs). Be aware that a single plate may contain extrachromosomal arrays as well as integrated lines.