Help: SP6/T7 specificity

[brett]

>How specific are SP6 and T7 RNA polymerase concering the other
>promoter sequences?
>
>I use RNA trancripts as in situ probes, generated from an cDNA
>with flanking T7 and SP6 promoters. The antisense probe gives
>me a very strong signal in the expected cell population. However,
>also the sense probe gives a signal in the same cells, although 
>much much weaker.
>I inerpreted this that by generating my sense probe with SP6,
>also some transcripts are formed from the T7 site and producing.
>
>Has anyone made similar observations? 
>
> 

Well, you may get a very small % of incorporation if you use the wrong enzyme,
but this will be negligible. A more likely explanation is that you are
seeing asymetric transcripts, which are more prominent when 3' overhangs
are used for linearization (I presume these are run-off transcriptions).
The solution is to use a 5' overhang or blunt. This is described in the
early SP6 RNAP literature.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu