Bio-Rad PDS-1000 / He™ and Hepta™ Systems User Manual
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Coating Washed Microcarriers with DNA
The following procedure is sufficient for six bombardments; if fewer bombardments are
needed, adjust the quantities accordingly.
Vortex the microcarriers prepared in 50% glycerol (30 mg/ml) for 5 minutes on a platform
vortexer to resuspend and disrupt agglomerated particles.
When removing aliquots of microcarriers, it is important to continuously vortex the tube
containing the microcarriers to maximize uniform sampling. When pipetting aliquots, hold the
microcentrifuge tube firmly at the top while continually vortexing the base of the tube.
Remove 50 µl (3 mg) of microcarriers to a 1.5 ml microcentrifuge tube.
Continuous agitation of the microcarriers is needed for uniform DNA precipitation onto
microcarriers. For added convenience and/or multiple samples, use a platform attachment on
your vortex mixer for holding microcentrifuge tubes.
While vortexing vigorously, add in order:
• 5 µl DNA (1 µg/µl)
• 50 µl 2.5 M CaCl
2
• 20 µl 0.1 M spermidine (free base, tissue culture grade)
Continue vortexing for 2–3 minutes.
Allow the microcarriers to settle for 1 minute.
Pellet microcarriers by spinning for 2 seconds in a microfuge.
Remove the liquid and discard.
Add 140 µl of 70% ethanol (HPLC or spectrophotometric grade).
Remove the liquid and discard.
Add 140 µl of 100% ethanol.
Remove the liquid and discard.
Add 48 µl of 100% ethanol.
Gently resuspend the pellet by tapping the side of the tube several times, and then by vor-
texing at low speed for 2–3 seconds.
4.2 Performing a Bombardment
Quick Guide (This summary is repeated in Section 8.4 as a tear-out sheet.)
Before the Bombardment
1. Select/adjust bombardment parameters for gap distance between rupture disk retaining cap
and microcarrier launch assembly. Placement of stopping screen support in proper posi-
tion inside fixed nest of microcarrier launch assembly
2. Check helium supply (200 psi in excess of desired rupture pressure).
3. Clean/sterilize:
Equipment: rupture disk retaining cap, microcarrier launch assembly
Consumables: macrocarriers/macrocarrier holders
4. Wash microcarriers and resuspend in 50% glycerol
5. Coat microcarriers with DNA and load onto sterile macrocarrier/macrocarrier holder the
day of experiment
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