( d. Pellet the microparticles by sp...)Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System

8. Add sterile 50% glycerol to bring the microparticle concentration to 60 mg/ml (assume no loss during preparation).

9. Store the microparticles at room temperature for up to 2 weeks.

Appendix 2. Coating DNA onto microcarriers
The following procedure is sufficient for six bombardments; if fewer bombardments are needed, prepare enough microcarriers for three bombardments by reducing all volumes by onehalf. When removing aliquots of microcarriers, it is important to vortex the tube containing the microcarriers continuously in order to maximize uniform sampling.

1. Vortex the microcarriers prepared in 50% glycerol (60 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles.

2. Remove 50 ml (3 mg) of microcarriers to a 1.5 ml microfuge tube.

3. While vortexing vigorously, add in order:
5 ml DNA (1 mg/ml)
50 ml CaCl₂ (2.5 M)
20 ml spermidine (0.1 M)

4. Continue vortexing for 2-3 minutes.

5. Allow the microcarriers to settle for 1 minute.

6. Pellet the microcarriers by spinning for 2 seconds in a microfuge.

7. Remove the liquid and discard.

8. Add 140 ml of 70% ethanol without disturbing the pellet.

9. Remove the liquid and discard.

10. Add 140 ml of 100% ethanol without disturbing the pellet.

11. Remove the liquid and discard.

12. Add 48 ml of 100% ethanol.

13. Gently resuspend the pellet by tapping the side of the tube several times, and then by vortexing at low speed for 2-3 seconds.

14. Remove six 6 ml aliquots of
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