BIOL309 Lecture Notes - Lecture 1: Dot Blot, Reporter Gene, Transgene
Document Summary
Nuclear run-on: measuring transcript abundance in nucleus: only see contribution of promoter activity because degradation takes place in the cytosol, way to monitor transcript stability is done by nuclear run-on (not used a lot because challenging) Requires isolation of nuclei from cells expressing the transcript. Chemically inhibit new transcription initiation but allow all transcripts that are underway to be completed. Incubate nuclei in the presence of 32p-utp (labels) Use this pool of mrna as a probe for a dot blot of specific genes. Regulation does not take place in the promoter. Transcriptional fusion: promoter::reporter gene fusion, reporter gene encodes product that we can easily detect and measure, by replacing coding gene with coding gene for reporter gene, the promoter"s activity can easily be monitored. Introducing it into a wild-type organism (still has the functional gene: transformants are not mutant for the transgene (transcriptional fusion gene, used in different experiments:
