Creativity

Innovation

Originality

Imagination

 

Salient

Salient is an excellent design with a fresh approach for the ever-changing Web. Integrated with Gantry 5, it is infinitely customizable, incredibly powerful, and remarkably simple.

Download

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&degC. 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.