| HOME >> BIOLOGY >> TECHNOLOGY |
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
'"/>
Source: