Extra Bands in in vitro transcription/primer extension
mytelka at zenith.Berkeley.EDU
Sun Sep 19 19:19:25 EST 1993
Whenever I do in vitro transcription followed by primer extension,
I seem to get a bunch of bands scattered both above and below the
expected band. These must represent in vitro transcription
artifacts, since I get clean results when I primer extend in vivo RNA.
I also get clean results when I run the in vivo RNA through the in
vitro transcription procedure, so it isn't my reagents degrading the
RNA. Any clues?
In vitro Transcription:
Template (various, generally QIAGEN column purified, but I've tried
CsCl banded. All should be DS supercoiled, single promoter.)
RNA Polymerase (generally E. coli, also T7)
DEPC treated water (and salts)
Incubate at 37 or 30 degrees
Phenol/Chloroform Extract, chloroform extract, precipitate with
NaOAc, wash, dry, resuspend, primer extend.
I'm looking for about a 170nt transcript.
Daniel Mytelka (mytelka at mendel.berkeley.edu)
More information about the Methods