Materials

1. Purified, endotoxin-free plasmid DNA (see Note 3).

2. Morpholino AS-ODNs prepared in cell culture-grade water and filter sterilized (AVI BioPharma, Corvallis, OR).

4. Tissue culture biosafety cabinet.

5. Adherent HeLa cell cultures (see Note 4).

6. Cell culture medium components: Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 IU/mL of penicillin, 100 ^g/mL of streptomycin; trypsin (0.25%)-EDTA (0.1%) solution; Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium (Mediatech).

7. Sterile plasticware for passaging cells (T-175 flasks), for plating assays (96-well plates), for cell pelleting (250-mL conical, polypropylene), and for dilutions (5.0-mL polypropylene round-bottomed tubes) (Falcon).

8. Tabletop centrifuge for pelleting cells (Beckmann Allegra 25R).

9. Gene Pulser II Electroporator with capacitance extender and 0.4-cm gene pulser cuvets (Bio-Rad, Hercules, CA).

10. MTT assay reagents: MTT, N,N-dimethylformamide (NN-DMF), sodium dodecyl sulfate, PBS (with Mg and Ca).

11. Shaker unit with microplate platform (Labline).

12. Microplate reader equipped with a 570 nM filter (MRX; Dynex).

13. Microplate injectable luminometer (Dynex MLX luminometer).

14. Microlite 1+ 96-well round-bottomed white microtiter plates (Dynex).

15. Dual-Luciferase Assay Kitâ„¢ (Promega, Madison, WI).

0 0

Post a comment