Editing Adenovirus Invitrogen (section)

== Transfection  ==

1. The day before transfection, trypsinize and count the 293A cells, plating them at 5 x 10<sup>5</sup> cells per well in a 6-well plate. Plate cells in 2 ml of normal growth medium containing serum. 

2. On the day of transfection, remove the culture medium from the 293A cells and replace with 1.5 ml of normal growth medium containing serum. Do not include antibiotics. 

3. Prepare DNA-GeneExpressoMax complexes for each transfection sample by performing the following: 

â¢ Dilute 1 ÎŒg of Pac I-digested pAd-DEST expression plasmid DNA in 250 ÎŒl of DMEM without serum. Mix gently. 

â¢ Mix GeneExpressoMax gently before use, then dilute 3 ÎŒl in 250 ÎŒl of DMEM medium without serum. Mix gently. 

â¢ Combine the diluted DNA with the diluted GeneExpressoMax. Mix gently. 

â¢ Incubate for 20 minutes at room temperature to allow the DNA-GeneExpressoMax complexes to form. 

4. Add the DNA-GeneExpressoMax complexes dropwise to each well. Mix gently by rocking the plate back and forth. Incubate the cells overnight at 37°C in a CO<sub>2</sub> incubator.<br>5. 48 hours after transfection, remove the medium containing the DNA-GeneExpressoMax complexes and replace with complete culture medium (i.e. D-MEM containing 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin). 

6. 48 hours post-transfection, trypsinize cells and transfer the contents of each well to a sterile 10 cm tissue culture plate containing 10 ml of complete culture medium. 

Caution: Remember that you are working with infectious virus at this stage and in all subsequent procedures. Follow the recommended guidelines for working with BL-2 organisms (see page 5 for more information). 

7. Replace culture medium with fresh, complete culture medium every 2-3 days until visible regions of cytopathic effect (CPE) are observed (typically 7-10 days post-transfection). For an example, see the next page. 

8. Replenish culture medium and allow infections to proceed until approximately 80% CPE is observed (typically 10-13 days post-transfection). 

9. Harvest adenovirus-containing cells by squirting cells off the plate with a 10 ml tissue culture pipette. Transfer cells and media to a sterile, 15 ml, capped tube.