Editing Adenovirus pAd52 (section)

== Generation of Recombinant Adenovirus GeneExpressoMax Protocol ==

1. Grow 293A cells in 6 well plate&nbsp; to be about 70-80% confluent at time of use (1 to 2 days) using DMEM + 10% bovine serum. <br>

2. Add plasmid DNA (2-5 μg/per well of each plasmid) to 100 μl DMEM without serum, mix well—&gt; Solution A.

3. Add 6 to 10 μl GeneExpressoMax to 100 μl DMEM without serum, mix well–&gt; Solution B.

4. Mix Solution A and B, incubate at room temperature for 20 min.

5. Add mixture to adherent 293 cell cultures dropwise around the dish without removing the medium.

6. Incubate at 37oC in a CO2 incubator for 2 days.

7. Aspirate medium and overlay with 3 ml F11 + 5% Horse Serum + 0.5% agarose* or refeed with 2 ml F11 + 5% Horse Serum**.

8. Incubate at 37oC. Plaques or CPE should appear after about 5 days. Plaques can be isolated for expansion by 10 days. Note that some vectors may replicate slowly and in such cases an additional 5 ml overlay can be added to the dishes at 10 days to allow for further plaque development.

Notes<br>* Prepare by mixing equal volumes of DMEM + 10% Horse Serum + Pen-Strep + L-Glutamine + 0.2% Yeast Extract and 1% Agarose. (If contamination with molds is an occasional problem fungizone can be added.)<br>** It is good laboratory practice to use plaque purified virus prior to extensive experimentation (i.e. use an agar overlay at step 8) but recombinant virus can also be recovered by incubation of transfected cells under liquid medium. Cpe can usually be detected by 5 days and should be complete by 7-8 days.<br>