Bio-Rad PDS-1000 / He™ and Hepta™ Systems - Manual (Page 24)

Fig. 4.4A and 4.4B. Insertion of macrocarrier into macrocarrier holder with plastic insertion tool.
3. Stopping screens
Transfer selected stopping screens (Figure 4.3) to individual Petri dishes for easier han-
dling. Sterilization by autoclaving is recommended. Alternatively, these parts can be ster-
ilized by soaking in 70% ethanol, followed by drying in a sterile environment.
4. Microcarriers
The following procedure prepares tungsten or gold microcarriers for 120 bombardments
using 500 µg of the microcarrier per bombardment, based on the method of Sanford, et
al.
[Methods in Enzymology, 217, 482-509 (1993)].
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 (a platform vortexer is useful).
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 concen-
tration 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.
Store dry tungsten and gold microcarriers in a dry, non-oxidizing environment to mini-
mize agglomeration.
21
Macrocarrier
Insertion
Tool
Helium
Macrocarrier
Helium
Macrocarrier
Holder
Direction of Tool
Movement