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. 2008 Aug 29;3(8):e3084. doi: 10.1371/journal.pone.0003084

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Gonçalves et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Extrachromosomal DNA was extracted from PER.tTA.Cre76 cells co-transfected with the rAAV vector shuttle plasmid pAAV.DsRed and the FLP-activatable rep68 expression plasmid pGS.pA+.Rep68 and infected 24 h later with Ad.floxedΨ.F50 alone (lane 1) or with Ad.floxedΨ.F50 plus hcAd.FLPe.F50 (lane 2). Positive (lane 3) and negative (lane 4) controls consisted of extrachromosomal DNA extracted from Ad.floxedΨ.F50- and mock-infected PER.tTA.Cre76 cells, respectively, co-transfected with pAAV.DsRed and the constitutive rep78/68 expression plasmid pGAPDH.Rep78/68. The extracts were treated with DpnI to selectively digest input, non-replicated, prokaryotic DNA and were subjected to Southern blot analysis using a probe corresponding to the DsRed.T4 ORF. Lane M, GeneRuler DNA Ladder Mix molecular weight marker (Fermentas). The positions and sizes (in kb) of the rAAV replicative intermediates (i.e., DMs and DDs) are indicated. The numerals at the left correspond to restriction DNA fragment sizes in kb.