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Transformation of Nematodes With the Helios Gene Gun

suspension can be stored for up to 2 months at -20 C (if not directly used for cartridge preparation).

USING THE TUBING PREP STATION
1. Clean the inner surface of the Tefzel tubing (external size 10 mm; internal diameter 6 mm; Bio-Rad) with 5 ml of absolute ethanol and dry with nitrogen (grade 4.8; 99.998% pure) in the rotating tubing holder for at least 5 minutes.

2. Sonicate the DNA-coated particle suspension in ethanol for 3 seconds (to achieve a homogeneous solution) and load it into the tubing at a rate of 5.56 ml/min using a syringe.

3. Allow the DNA-coated particles to settle for 3 minutes.

4. Remove the ethanol solution quickly (within 30 seconds).

5. For drying the wall-attached particles, flush nitrogen gas through the tubing for 3 minutes at a flow rate of 0.35 l/min.

6. Finally, cut the resulting particle-covered tubing into individual cartridges 16 mm in length (one full length of tubing results in nearly 50 cartridges) to fit into the 12-shot barrels of the Helios gene gun (Bio-Rad). See Figure 2.

7. In a desiccated atmosphere, these can be stored at 4 C for up to 8 months without losing activity.

8. Deliver the DNA-coated gold particles using 4 blasts of helium (grade 4.5; 99.995% pure) applied to the cartridges at a pressure of 300 p.s.i. and a distance of 3 cm to the target (in the case of C. elegans).

9. A pressure of 120180 p.s.i. at a distance of 2 cm using 1 blast of helium is useful for the transfer into parasitic nematode L. sigmodontis. After this procedure, the female parasites may be cultivated at 37 C for up to 2 weeks in RPMI medium (5% CO2).

SELECTION FOR TRANSFORMED TRAITS OF C. ELEGANS

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