Particle bombardment of Arabidopsis leaves

Gold Preparation

  1. Make a suspension of 50mg gold particles (1.0 mm) in 1mL sterile dH2O
  2. Vortex the suspension vigorously for 1-2 min
  3. Centrifuge the suspension at 13 000 g for 1 min
  4. Discard supernatant
  5. Wash the gold particles three times with 100% ethanol by vortexing followed by centrifuging
  6. Wash once with sterile dH2O – vortex, centrifuge and discard the supernatant
  7. Resuspend particles in 1 mL sterile dH2O and aliquot 25 mL per 1.5 mL eppendorf tube.  You will need to continuously vortex the suspension as you aliquot
  8. Store tubes at 4 oC

Coating the gold particles with Plasmid DNA

  1. Resuspend a 25 mL aliquot of gold particles by vortexing or with several pulses in a sonicator (a water bath Ultrasonic Cleaning Device)
  2. Add 2-5 mg (up to 5mL volume) of plasmid DNA to the gold suspension and vortex or sonicate briefly again
  3. Add 25mL 2.5 M CaCl2 and pipette up and down 5-10 times (do not cause bubbles).  Vortex briefly.  This step precipitates the DNA on to the gold
  4. Add 10 mL of 0.1 M spermidine, mix by pipetting and vortex at low speed for 2 min.  Incubate the tube on ice for 5-30 min.  Spermidine protects the DNA from cellular DNAses
  5. Pellet the DNA-coated gold particles by centrifuging for about 10s
  6. Remove supernatant and add 180 mL of 100% ethanol.  Resuspend the gold particles by pipetting up and down followed by sonicating.  This is a washing step
  7. Pellet the gold particles again and discard the supernatant
  8. Resuspend the particles in 100 mL 100% ethanol, mix and sonicate to fully resuspend.  The particles can be stored at -20 oC for several weeks

Operating the Gene Gun

  1. Place a 5 mL aliquot of your gold preparation on the downward facing surface of the microcarrier
  2. Assemble the microcarrier launch assembly, placing a stopping screen at the  base (do not use a microcarrier for the first blank shot).  Place the microcarrier launch in the brass cylinder, position the microcarrier in the top and position the shelf in the top position
  3. Open the main valve on the helium cylinder and adjust the pressure to 1100 psi if necessary
  4. Place a rupture disk (1100psi) in the cap and screw into position, tighten the cap with the lever
  5. Place the sample on the sample tray and slot in to the desired shelf position
  6. Close the chamber and turn on the vacuum pump (hold VAC).  When the vacuum gauge needle will go no further (usually about 28) flick the switch to HOLD
  7. Press the fire button (hold) and watch the pressure gauge on the top of the gun rise.  When the pressure reaches 1100psi the rupture disk will burst
  8. After bombardment is complete release the vacuum from the chamber by pushing the vacuum button to VENT
  9. When finished, close main valve of the helium cylinder and release the helium pressure by repeatedly firing the gun until the cylinder shows the pressure has returned to zero.  Turn off the gene gun and the vacuum pump

Plant Material

We bombard the lower epidermis of 5-6 week-old, fully expanded, source leaves of short-day grown Arabidopsis.

Under our conditions, bombardment works optimally at 1100psi with the leaves placed on the floor or lower shelf of the gun.