Notes: These authors performed gel shift (EMSA) assays to determine whether purified VirA binds to the vapA promoter. Radiolabeled DNA fragments for the EMSA assays were prepared using DNA Polymerase I Large (Klenow) Fragment. Primer extension using ImProm-II™ Reverse Transcriptase localized the transcription start site within the vapA promoter. To characterize the transcriptional organization of the virR gene cluster, the authors performed reverse transcription using ImProm-II™ Reverse Transcriptase and Random Primers, followed by PCR using a combinations of primers in opposite orientations throughout the gene cluster. The plasmids used in this study were purified by alkaline lysis method or using the Wizard^® Plus SV Minipreps DNA Purification System.
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