T7 transcription yields

unknown at dl.ac.uk unknown at dl.ac.uk
Fri Dec 31 00:25:57 EST 1993


Hello, thought I'd share some experience about in vitro transcription for
the recent request to do so. I find that the key consideration is to start
with the highest quality template possible. While I get good yields from
Qiagen preps, these transcripts sometimes don't transfect mammalian cells
as well as CsCl prepped template. So, depending on what you have in store
for these transcripts, prepare your templates as you see fit. Also, the
use of pyrophosphatase (sequencing grade - USB (aside, anyone know of another
supplier of this enzyme?)) will favor the forward reaction and allow for
longer incubation times using T7 RNA pol. However, I have not yet seen a
difference when using pyrophosphatase in SP6 reactions. Any clues? Finally,
in the interest of RNAse paranoia, it is wise to add RNASin (first add DTT!)
to prevent degradation. Good luck, and let me know if there are other
suggestions.

Brett Lindenbach
brett at borcim.wustl.edu



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