Measuring transcription in vitro:

Background / Usage:

Broccoli and Spinach are two versions of an RNA aptamer tag that fluoresces with similar spectral properties to GFP when properly folded. Both require a small-molecule fluorophore (DFHBI); the Barrick lab has a stock of fluorophore, and it can be purchased from a number of vendors (as of 5/2017). These aptamers can be used for a variety of applications; this protocol covers using them to measure transcriptional output from T7 RNA Polymerase / promoter mutants in vitro, but they can also be used to measure transcription in vivo in bacteria or eukaryotic cells or (by using the aptamer as a fusion tag) to determine subcellular localization of RNA transcripts.

For in vitro experiments, we have had most success using linear templates ~120bp. These can be produced either by subjecting overlapping oligos to an extension reaction, or by using shorter primers to amplify the template from a plasmid. Note that both of these methods can be used to swap promoters if needed.

Designing and Amplifying Templates

in vitro Transcription Assay

-- Main.ColinBrown - 25 May 2017

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Topic revision: r1 - 25 May 2017 - 19:57:09 - Main.ColinBrown
 
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