(r1) Barrick Lab :: ProtocolsBroccoliAptamerInVitroAssays

---++ 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 <em>in vivo </em>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

---+++ <em>in vitro</em> Transcription Assay 
 

-- Main.ColinBrown - 25 May 2017

Barrick Lab > ProtocolList > ProtocolsBroccoliAptamerInVitroAssays

Contributors to this topic

ColinBrown

Topic revision: r1 - 2017-05-25 - 19:57:09 - Main.ColinBrown