Extra Bands in in vitro transcription/primer extension

Daniel Mytelka 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)
Appropriate salts
1mM+  nucleotides
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)

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