PROTOCOLS       

Microparticle Bombardment Protocol

Unc-119 Worm Preparation

  1. Take one 5X 10 cm plate for each bombardment and place it in 37°C incubator with the lid slightly ajar. It is imperative that you dry out the bombardment plates as long as possible so that the M9-worm mixture that will be added later will soak in.
  2. Take out egg plates from 20°C incubator. The unc-119 worms should be clustered in many small patches and most if not all of the egg solution should have been eaten. Make sure that there is no mold growing on the surface of the plates – if there are small patches of mold, try to excise them from the surface of the plate with a sterile spatula.
  3. Add 10 mL of M9 to each egg plate. Use sterile disposable plastic spreader to dislodge worms from surface of plate. Pipet worm-M9 liquid into 50 mL labeled conical tube (one tube for each set of 4 plates). Wash worms off surface of egg plate with 10 mL M9 again – spreader is not necessary for 2nd wash.
  4. Spin down worms for 2 minutes at 1500 RPM.
  5. Take off supernatant with vacuum filter or pipet.Add 25 mL of M9 to each tube containing worm pellet. Mix by hand to resuspend. Let the worms settle by gravitation. This will separate larvae and small particles from the adult worms. Set a timer for 25 minutes. After 25 minutes, go to step 14.

DNA/Gold Preparationn

  1. Transfer DNA plasmids to be bombarded to siliconized tubes. Volume of DNA should be 50 mL and approximately 10 mg. Volume up to 200 mL may be used in order to achieve the 10 mg concentration. Take spermadine solution out of –80°C incubator – when it thaws, place on ice. Place 2.5 M CaCl2 in 37°C waterbath.
  2. Vortex gold solution vigorously to resuspend particles. Pipet up and down with P200. Add 100 mL of gold solution to each bombardment plasmid. Vortex for 1 minute.
  3. Add 150 mL of 2.5M CaCl2 solution to each bombardment tube. Vortex for 1 minute. If the volume of your DNA is more than 50mL, you must adjust the volume of 2.5M CaCl2 accordingly. For example, for a 100 mL volume, add 300 mL CaCl2.
  4. Add 60 mL of spermadine to each bombardment tube. Vortex for 5 minutes. If the volume of your DNA is more than 50mL, you must adjust the volume of spermadine accordingly. For example, for a 100 mL volume, add 180 mL spermadine.
  5. Tap-spin bombardment tubes – pulse up to 6000 RPM on tabletop centrifuge, take off supernatant. The pellet will be tight, but be careful not to disturb it with the pipet tip.
  6. Add 300 mL 70% ethanol to bombardment tubes. Vortex for 5 minutes.
  7. Tap-spin bombardment tubes, remove supernatant.
  8. Add 170 mL 100% ethanol to bombardment tubes. Vortex on full speed for 10 minutes. After 10 minutes, turn vortex down to shake. Keep vortex on shake until ready to bombard DNA.

Bombardment Plate Preparationn

  1. Take off M9 supernatant. It is important to remove as much liquid as possible at this step – losing a small portion of the worms is preferable to having too much liquid left.
  2. Fill one container with ice for each bombardment plate. Place Saran wrap over the ice to prevent contamination. This also keeps the exterior of the plates from getting too wet. Add one tube of gravitated worms to each bombardment plate. Try to spread out the worms evenly across the plate by tilting it from side to side. Let the worms sit on ice for at least half an hour. Keeping the worms on ice prevents them from clustering together.

Macrocarrier Preparationn

  1. Add Dryerite to one 150cm Petri dish for each bombardment. Add enough Dryerite to cover approximately 20% of the dish surface.
  2. Fold a Kimwipe in half and place it on top of the Dryerite.
  3. Dip 7 macrocarriers in 95% ethanol and then place them on top of the Kimwipe making sure that they do not touch the dish surface or the Dryerite.
  4. Place the lid on the dish slightly ajar so that the ethanol can evaporate.

Test Fire of Bombardment Chamberr

  1. Place a rupture disc in the rupture disc holder. Use a rupture disc with pressure limit equal to the discs that you plan to use for the actual DNA bombardments.
  2. Screw the rupture disc holder into the bombardment chamber’s gas acceleration tube until it cannot turn anymore. Use the torque wrench (silver metal cylinder with thicker black cylinder around it at one end) to tighten the rupture disc holder.
  3. Close the bombardment chamber door.
  4. Turn on bombardment apparatus (left most button to On setting).
  5. Turn on vacuum pump.
  6. Switch middle button up to “Vac” setting. Allow pressure to reach 28 inches Hg. Press middle button all the way down (two clicks) to “Hold.”
  7. Turn on gas. Adjust the pressure gauge to 200 psi higher than the pressure limit of your rupture disc. For example, a 1800 psi rupture disc would require 2000 psi of pressure for proper rupturing.
  8. Press and hold rightmost button marked “Fire.” Let the pressure increase until a loud popping sound is heard. Release “Fire” button to allow He pressure to diminish. At the same time, switch central button to “Vent” (one click up from “Hold”). If no popping sound is heard when He Pressure Gauge reads at least 200 psi above rupture disc pressure limit, it most likely means that more than one rupture disc has been inserted into the rupture disc holder. The rupture discs have a tendency to stick together and it is important to make sure that only one disc is used at a time. If the disc does not rupture, use the torque wrench to slowly release the pressure. Try the test fire again with a fresh rupture disc.
  9. Unscrew the top part of the rupture disc holder from the bottom part. Use tweezers to take out broken rupture disc.

Bombardment of DNA Plasmids

  1. Clean surface of macrocarrier holder with a Kimwipe sprayed with ethanol.
  2. Add one dry macrocarrier to each hole in the macrocarrier holder. Use the red plastic tool to secure the macrocarriers by placing it on top, pushing and rotating in small circles. The macrocarriers will make a crinkling sound as they are being screwed in place. It is important to do steps 29-31 before cleaning the other equipment because it takes approximately 20 minutes for the DNA/gold mixture to dry out.
  3. Vortex prepared DNA sample on full speed briefly to resuspend gold particles. Pipet up and down and scrape the sides of the tube with the pipet tip to free as many gold particles as possible. Add 20 mL of DNA/gold mixture to the center of each macrocarrier. Use the remaining liquid in the tube to even out the amount of DNA/gold mixture in each macrocarrier. If there is a significant amount of gold left on the sides of the tube, add 20 mL of 100% ethanol and vortex to resuspend. Add the remaining DNA/gold mixture to the macrocarriers evenly.
  4. Wipe down the rest of the equipment (both parts of rupture disc holder, stopping screen holder, macrocarrier launch assembly, target shelf) and the bombardment chamber interior with an ethanol-sprayed Kimwipe.
  5. Place a stopping screen on the two prongs of the stopping screen holder. Place the stopping screen holder into the macrocarrier launch assembly.
  6. Dip a rupture disc of the desired pressure limit in ethanol briefly and place in the rupture disc holder (as before in the test fire). Screw in the rupture disc holder tightly.
  7. When the macrocarriers and the DNA/gold mixture have dried out, invert the macrocarrier holder and place it on top of the stopping screen holder. Rotate the lever on the stopping screen holder so that the two pieces are locked in place in the launch assembly.
  8. Slide the macrocarrier holder unit into the second shelf slot of the bombardment chamber so that the curved edges of launch assembly face in and the handle on the stopping screen holder faces out. Turn the handle until the holes in the stopping screen holder line up with the tubes of the hepta adapter (the lower half of the rupture disc holder).
  9. Wipe down the bottom of the prepared unc-119 worm plate so that it will not slide around. Place two pieces of double-stick tape on the target shelf and make sure that the worm plate stays on the target shelf firmly.
  10. Slide the target shelf into the bottom shelf slot. Take off the Petri dish cover.
  11. Close bombardment chamber door.
  12. Turn on vacuum pump.
  13. Switch middle button all the way up to “Vac” until vacuum pressure reaches 28 inches Hg. Switch middle button all the way down to “Hold.”
  14. Turn on He gas. Make sure that pressure is adjusted to 200 psi higher than rupture disc pressure limit.
  15. Press and hold rightmost “Fire” button until rupture disc pops (200 psi higher than rupture disc pressure limit). Release “Fire” button. Switch middle button to “Vent.” Remove bombarded worm plate.

Washing Bombarded Worms

  1. Label 20 plates with DNA plasmid name (or abbreviation) for each bombardment.
  2. Add 10.5 mL of M9 to each bombardment plate. Gently swirl M9 so that unc-119 worms dislodge from plate. Take off M9 with pipet.
  3. Add ~ 0.5 mL to each labeled plate. The first few milliliters of the worm mixture will have more worms than the last few milliliters, so often it’s best to add a little less liquid to the first few plates and the extra liquid to the last plates.
  4. Place the 21 5X Peptone plates (the 20 new plates and the original bombardment plate) in the 25°C incubator for 2 weeks.