University of Georgia

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Parrott Lab

Procedure for microparticle/helium bombardment

Materials

  • Biolistic particle delivery system PDS-1000/He
  • Microcarriers: 0.6 micron gold (Au) Bio-Rad, Cat. No. 165-2263
  • Macrocarriers: Bio-Rad, Cat. No. 165-2335
  • Rupture Disks: 1100 psi. Bio-Rad, Cat. No. 165-2329
  • PDS-1000/He stop screens: Bio-Rad, Cat. No. 165-2336
  • Eppendorf tubes
  • Eppendorf centrifuge
  • Sonicator
  • EtOH 100% (absolute)
  • H2O ultrapure, sterile
  • CaCl2 2.5 M sterile (ice cold)
  • Spermidine 0.1 M sterile (ice cold)
  • Pipetman and tips for 20, 200, and 1000 μl
  • Forceps
  • Timer
  • Nescofilm
  • Marker
  • Sterile petri dishes

Preparation of microcarriers

  • Prepare gold before use, do not store
  • Place 10 mg of gold powder (microcarriers) in eppendorf tube and add 1 ml 100% EtOH.
  • Sonicate 10 sec. & place the tube on ice for 30 sec.
  • Repeat three times
  • Centrifuge 5 min at 7000 rpm.
  • Remove the EtOH (do not remove the metal pellet).
  • Add 175μl of EtOH to the 10 mg of gold powder.
  • Vortex 1 min.
  • Sonicate the suspension 10 to 15 seconds, twice.
  • Withdraw a 35 μl aliquot before the microcarriers settle.
  • Transfer it to a sterile 1.5 ml Eppendorf tube.
  • Before withdrawing any other aliquot, vortex and sonicate as above.
  • Centrifuge all the aliquots briefly (about 10 sec) at 10,000 rpm.
  • Remove the supernatant.

The rest of the protocol is for one aliquot (used for three bombardments):

  • Add 1 ml of sterile H2O (do not resuspend)
  • Centrifuge for 5 min at 2000 rpm.
  • Remove H2O.
  • Add 25 μl of DNA (6 ng/μl) solution to the metal pellet.
  • Resuspend the microcarriers by pipetting up and down.
  • Vortex 2 to 3 sec.
  • Sonicate 10 to 15 sec.
  • Keep the tube vortexing while proceeding with other aliquots. Before starting the next step, sonicate the tube 10 to 15 sec.
  • Add 220μl of sterile H2O.
  • Resuspend by pipetting up and down.
  • Vortex 2 to 3 sec.
  • Sonicate 10 to 15 sec.
  • Keep vortexing while proceeding with the others. Before starting the next step, sonicate the tube 5 to 7 sec.
  • Add 250μl of 2.5 M CaCl2 (ice cold).
  • Resuspend by pipetting up and down.
  • Vortex 2 to 3 sec.
  • Sonicate 10 to 15 sec.
  • Keep vortexing while proceeding with the others.
  • Before starting the next step, sonicate the tube 5 to 7 sec.
  • Add 50μl of 0.1 M spermidine stock (ice cold).
  • Do not use spermidine stock that is more than three months old, if stored at -20�C.
  • Resuspend by pipetting up and down.
  • Vortex 2 to 3 sec.
  • Sonicate 10 to 15 sec.
  • Keep the tube vortexing while proceeding with others.
  • Place the tube on ice 2 to 3 min.
  • Vortex 10 min.
  • Centrifuge at 500-1000 rpm (lowest possible speed) for 5 min.
  • Remove supernatant. Do not let the pellet dry.
  • Resuspend DNA/microcarriers in 600 μl of 100% EtOH.
  • Vortex 2 to 3 sec.
  • Sonicate 7 to 10 sec.
  • Keep the tube vortexing while proceeding with others.
  • Centrifuge 5 min at 500-1000 rpm.
  • Remove EtOH.
  • Resuspend the pellet in 36 μl of 100% EtOH by flicking.
  • Sonicate 5 to 7 sec.
  • Leave tubes on ice 1 hour.
  • Resuspended the DNA/microcarrier suspension by pipetting up and down with a 20 μl pipette until evenly resuspended.
  • Sonicate 10-15 seconds.
  • Withdraw 10 μl of the resuspended DNA/microcarriers and dispense evenly onto the surface of the macrocarrier (one tube is enough for three macrocarriers).
  • Allow the EtOH to dry completely.
  • The surface of the macrocarrier should be covered with gold evenly.