Bio-Rad Helios® Gene Gun System - Manual (Page 25)

Loading the DNA/Microcarrier Suspension into Gold-Coat Tubing Using the
Tubing Prep Station
Materials
Supplied
Tubing Prep Station (see Section 4.3)
Gold-Coat tubing
To be Supplied by User
Microcarrier/DNA suspension(s) from Section 5.1, Precipitation of DNA onto
Microcarriers, at room temperature
Ultrasonic cleaner
Vortexer
100% ethanol
Peristaltic pump
Minute timer
Nitrogen tank (see Section 2.4)
Nitrogen regulator (see Section 2.4)
Scissors
Procedure
Time considerations: Since only one piece of Gold-Coat tubing can be coated at a time,
this procedure may take 15–30 min for the first sample and 15–20 min for additional samples.
1. Set up the Tubing Prep Station and connect to a nitrogen tank as described in Section 4.3.
2. Prior to using the Tubing Prep Station, a peristaltic pump should be set up and calibrat-
ed to be used later for removing ethanol from the tubing at the rate of 0.5–1.0"/sec.
[Note: 1 ml of liquid occupies 8.5" (21.5 cm) of tubing, removing liquid at 0.5–1.0"/sec
is the same as 0.06–0.12 ml/sec or 3.6–7.2 ml/min.] Using a 10 ml graduated cylinder, cal-
ibrate a peristaltic pump at 5.5–6.0 ml/min. The tubing at the end of the peristaltic pump
which will be connected to the Gold-Coat tubing should have an inside diameter of 1/8".
3. If a peristaltic pump is not available, a syringe fitted with a 16–18" piece of silicone adap-
tor tubing and inserted into the syringe sleeve and clamped onto the base of the Tubing Prep
Station may be used to remove the liquid from the Gold-Coat tubing (see Section 4.3).
Liquid should be removed from the Tefzel tubing at 0.5–1.0"/sec. Practice removing liq-
uid 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 which 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 Section 8.2]).
4. Prior to preparing cartridges, ensure that the Gold-Coat tubing is completely dry by purg-
ing 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 tub-
ing 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 Gold-Coat tubing for at least 15 min immediately prior
to using it in the following steps.
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