BIO RAD Helios Gene Gun System Instruction Manual page 26

Section 5 Operation of the Helios Gene Gun System
− Purified plasmid DNA resuspended in distilled water or 10 mM Tris (pH 8.0), 1 mM EDTA
− Ultrasonic cleaner (for example, Fisher FS3, Branson 1210)
− Analytical balance capable of weighing microgram quantities
− Microcentrifuge
Procedure
Time considerations: preparation of the DNA/gold suspension requires approximately 30 min. Several
samples may be prepared simultaneously without a significant increase in time.
1. Prepare a stock solution of 20 mg/ml PVP in ethanol in a small screw-cap container. Dilute this solution
with ethanol to prepare PVP solutions at the desired concentration (generally 0.01–0.1 mg/ml); prepare
3.5 ml of the dilute solution for each 30" length of GoldCoat Tubing, (25" to be coated) in the Tubing
Prep Station. Keep these solutions tightly capped when not in use. Prepare solution daily.
2. In a 1.5 ml microcentrifuge tube, weigh out gold microcarriers. (Refer to Procedure 1 for a detailed
description on determining MLQ. Refer to Table 2 for suggestions on the relative amounts of gold and
microcarriers required and on the length of tubing produced.)
3. To the measured gold add 100 µl of 0.05 M spermidine. (However, if the volume of plasmid to be added
in step 5 is greater than 100 µl, refer to the discussion above for Procedure 2: Determining the DNA
Loading Rate, and add the appropriate volume of spermidine.)
4. Vortex the gold and spermidine mixture for a few seconds, then sonicate for 3–5 sec using an ultrasonic
cleaner to break up gold clumps.
5. To the gold and spermidine mixture, add the required volume of plasmid to achieve the desired DLR.
(Refer to Procedure 2 for a detailed description on determining DLR. Refer to Table 2 for suggestions
on the relative amounts of gold and microcarriers required and on the length of tubing produced.) For
cotransfection of multiple plasmids, add each of the plasmids at this step. DNA does not associate with
the microcarriers prior to addition of CaCl
6. Mix DNA, spermidine, and gold by vortexing ~5 sec.
7. While vortexing the mixture at a moderate rate on a variable-speed vortexer, add 100 µl of 1 M CaCl
dropwise to the mixture. The volume added should equal that of the spermidine added in Step 3.
8. Allow the mixture to precipitate at room temperature for 10 min.
9. Most of the gold will now be in the pellet, but some may be on the sides of the tube. The supernatant
should be relatively clear. Spin the microcarrier solution in a microcentrifuge ~15 sec to pellet the gold.
Remove and discard the supernatant.
10. Resuspend the pellet in the remaining supernatant by vortexing briefly. Wash the pellet three times with
1 ml of fresh 100% ethanol each time; spin ~5 sec in a microcentrifuge between each wash. Discard the
supernatants.
11. After the final ethanol wash, resuspend the pellet in 200 µl of the ethanol solution containing the
appropriate concentration of PVP prepared in step 1. Transfer this suspension to a 15 ml disposable
polypropylene centrifuge tube with a screw cap. Rinse the microcentrifuge tube once with 200 µl of the
same ethanol/PVP solution and add to the centrifuge tube. Add the necessary volume of the ethanol/
PVP solution to the centrifuge tube to bring the DNA/microcarrier solution to the desired MLQ.
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