Editing Adenovirus AdEasy (section)

== Generation of Recombinants in Bacterial Cells  ==

1. Prepare electrocompetent BJ5183 cells in 20 ml/tube aliquotes. 

2. Linearize the shuttle plasmids with PmeI. Usually one-fifth of a miniprep (typically 100-500 ng) is sufficient. After digestion, DNAs are phenol-chloroform extracted, ethanol precipitated, and resuspended in 6.0 ml H2O. 

3. Co-transform PmeI-digested shuttle plasmid with 1.0 ml adenoviral backbone vector (100 ng/ul). Twenty ul of electrocompetent E. coli BJ5183 cells were added and electroporation was performed in 2.0 mm cuvettes at 2,500V, 200 Ohms, and 25 mFD in a Bio-Rad Gene Pulser electroporator. Resuspend transformation mix in 500 ml L-broth (incubation at 37oC for 10-20 min is optional, depending on background, see FAQ's). Plate on 4 to 5 LB/kan plates, and grow at 37oC overnight (16-20 hrs). 

4. Pick up 10 to 20 smallest colonies, and grow them in 2 ml L-broth containing 25 mg/ml kanamycin for 10-15 hours. 

5. Perform minipreps using the conventional alkaline lysis method, and check the sizes of supercoiled plasmids by running one-fifth of a miniprep on 0.8% agarose gel. 

6. Restriciont digest DNA from clones with PacI. Candidate clones usually yield a large fragment (near 30 kb), plus a smaller fragment of 3.0kb or 4.5kb. 

7. Re-transform one microliter of the correct recombinant plasmid minipreps into DH10B (or your favorite plasmid propagation strain). Plasmids are purified by CsCl -banding (see Appendix D) or using commercially available purification kits for transfection of 293 cells.