BIO RAD Helios Gene Gun System Instruction Manual page 28

Section 5 Operation of the Helios Gene Gun System
Tefzel Tubing at 0.5–1.0"/sec. Practice removing liquid at this rate from the Tefzel Tubing by cutting a
~30" piece of tubing, loading it with ~3.0 ml of ethanol (~24" (~60 cm) of tubing), inserting it into the
Tubing Prep Station, and removing the liquid at 0.5–1.0"/sec. Marking the tubing at several points and
using a timer that measures time in seconds should make removal more accurate. It should take 25–45
sec to draw the liquid from the entire length of tubing. (Note: use ethanol rather than water for practicing
this step because if water leaks into the Tubing Prep Station, it may contaminate subsequent pieces of
Tefzel Tubing, leading to poorly coated tubes. See Cartridge Preparation in Section 8.
4. Prior to preparing cartridges, ensure that the GoldCoat Tubing is completely dry by purging with
nitrogen. Insert an uncut piece of tubing into the opening on the right side of the Tubing Prep Station.
The edge of the hole is beveled to permit easier insertion. Push the tubing into the hole and into the
tubing support cylinder. At the opposite end of the tubing support cylinder is an O-ring. There will be
slight resistance as the tubing is pushed into the O-ring; insert the tubing another 1/2" (1 cm).
5. Using the knob on the flowmeter, turn on the nitrogen and adjust the flow to 0.3–0.4 LPM. Allow
nitrogen to flow through the GoldCoat Tubing for at least 15 min immediately prior to using it in the
following steps.
6. Remove the GoldCoat Tubing from the Tubing Prep Station. Turn off the flow of nitrogen to the Tubing
Prep Station using the knob on the flowmeter.
7. From the dried GoldCoat Tubing cut a 29–30" (~75 cm) length of tubing for each 3 ml sample of
microcarrier/DNA suspension. (Note: Cutting the tubing with a scissors may distort the shape of the
end; the tubing may be easier to insert into the tubing support cylinder if the end is subsequently cut in
the tubing cutter.) Insert one end of the GoldCoat Tubing into the end of the adaptor tubing fitted to the
10 cc syringe (see step 5 of Setting Up the Tubing Prep Station, in Section 4).
8. Vortex the microcarrier suspension and, if necessary, sonicate briefly to achieve an even suspension
of gold. Invert the tube several times to resuspend the gold; immediately remove the cap and quickly
draw the gold suspension into the GoldCoat Tubing approximately 22–24" (~58 cm) (6–8" (~17 cm) from
the end). AVOID DRAWING BUBBLES INTO THE GOLDCOAT TUBING: Do not vortex the microcarrier
suspension while drawing it into the tubing. Do not try to remove all of the liquid from the container with
the suspension. Remove the tubing from the suspension and continue drawing the suspension into the
tubing another 2–3" (~6 cm) to leave some space at each end.
9. Immediately bring the GoldCoat Tubing to a horizontal position and slide the loaded tube, with
syringe attached, into the tubing support cylinder in the Tubing Prep Station until the tubing passes
through the O-ring.
10. Allow the microcarriers to settle for 3–5 min. Detach the GoldCoat Tubing from the adaptor tubing
and attach to the tubing on the peristaltic pump or on the 10 cc syringe. (Be careful not to rotate the
Gold-Coat tubing.) Remove ethanol at the rate of 0.5–1.0"/sec (this should require 30–45 sec).
11. Detach the peristaltic pump or syringe from the GoldCoat Tubing. Immediately turn the GoldCoat Tubing
180° while in the groove and allow the gold to coat the inside surface of the tubing for 3–4 sec.
12. Turn the switch on the Tubing Prep Station to ON (I) to start rotating the Tubing Prep Station.
(Warning: Keep objects away from the gears of the Tubing Prep Station while in operation.)
13. Allow the gold to smear in the tube for 20–30 sec, then open the valve on the flowmeter to allow
0.35–0.4 LPM of nitrogen to dry the GoldCoat Tubing while it continues to rotate.
14. Continue drying the GoldCoat Tubing while turning for 3–5 min.
24 | Helios Gene Gun System