in vitro transcription reactions

Randall Howard rfhoward at u.washington.edu
Sun Nov 27 03:36:38 EST 1994


Does anyone have advice about setting up eukaryotic in vitro transcription
reactions in a new system (I've not done any before)?  What are major
problem areas?  What's the best order of changes to try in optimizing
extract preparation and the reaction conditions and the best choice for
template/transcription product in the system?  By the way, can one get
enough signal from endogenous (genomic) gene if one is looking at a
regulated gene (say, regulated temporally or by external activator)? 
What's the "best" assay to use for the product?  I've seen labeled run-on
products, S1 nuclease production and primer extension all being used. 

Any advice would be appreciated.  Thanks in advance. 

Randy Howard
rfhoward at u.washington.edu




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