( tes. The surface of a densely ...)Transformation of Nematodes With the Helios Gene Gun

tes. The surface of a densely populated NGM agar plate is subsequently rinsed with tap water. A drop of water containing nematodes is placed in the center of a 6 cm Petri dish on ice (to reduce their motility). In this state C. elegans could be used for microparticle bombardment (Figure 1A).

For the same purpose adult females of L. sigmodontis must be isolated from the pleural cavity of their laboratory host Mastomys coucha (Wegerhof and Zahner, 1985), and stored in RPMI medium (supplemented with 10% FCS, Gibco-BRL; 1% Penicillin and Streptomycin). Single parasites are transferred to an agar plate and used for microprojectile bombardment.

CONDITIONS OF BOMBARDMENT
DNA-coated gold particles are chosen as microparticles following a modified Bio-Rad protocol for the Helios gene gun. The protocol is as follows:
1. Mix 50 mg of gold microparticles (Bio-Rad; a 1:1 mixture of particles 0.6 /1.0 in diameter) with 100 l of 0.05 M spermidine (Sigma), and sonicate 3 times for 3 seconds in an Eppendorf tube (1.5 ml).

2. Add 200 l of purified supercoiled plasmid DNA to the carrier suspension (in a total volume of 100 l). It is possible to use more than one plasmid simultaneously.

3. CaCl₂ (100 l, 1 M) is now added dropwise with continuous vortexing. Precipitation was allowed to take place for 10 minutes at room temperature.

4. Wash the suspension three times with 1 ml of absolute ethanol (Sigma-Aldrich), and pellet the sample by centrifugation (12,000 rpm for 1 minute, Heraeus Biofuge).

5. Finally, the pellet is resuspended in 3.5 ml of ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP; MW 360,000). In this state a DNA-coated gold particle
'"/>

Source:

Page: All 1 2 3 4 5 6 7
Related biology technology :

1. Highest Possible Transformation Efficiencies for High-Throughput Applications
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System
4. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
5. Transformation of Filamentous Fungi by Microprojectile Bombardment
6. Transformation of Filamentous Fungi by High-Voltage Electroporation
7. Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
8. Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun
9. Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment