Editing Adenovirus Invitrogen (section)

== Digestion with PacI  ==

Before transfecting pAd-DEST expression clone into 293A cells, the plasmid must be digested to expose the left and right viral ITRs to allow proper viral replication and packaging. Each pAd-DEST vector contains Pac I restriction sites (refer to the maps of each vector in the pAd/CMV/V5-DEST and pAd/PL-DEST manual for the location of the Pac I sites). Digestion of the vector with Pac I allows exposure of<br>the left and right viral ITRs and removal of the bacterial sequences (i.e. pUC origin and ampicillin resistance gene). Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. 

1. Digest at least 5 ÎŒg of purified plasmid DNA of your pAd-DEST expression construct with Pac I (New England Biolabs, Catalog no. R0547S). Follow the manufacturerâs instructions. 

2. Purify the digested plasmid DNA using phenol/chloroform extraction followed by ethanol precipitation or a Qiagen DNA purification kit <br> Note: Gel purification is not required.<br> <br>3. Resuspend or elute the purified plasmid, as appropriate in sterile water or TE Buffer, pH 8.0 to a final concentration of 0.1-3.0 ÎŒg/ÎŒl.