In vitro transcription with T7 RNAP

brett brett at BORCIM.WUSTL.EDU
Tue Mar 4 11:27:17 EST 1997


>Has anyone else faced problems with in vitro RNA transcription with T7 
>RNA polymerase? 
>
>I'm getting very faint or smeared RNA bands with the system. I've had 
>this problem before and it seems to be due to the impurity of the DNA 
>template. Of course it might be due to something else as well.
>
>I need to do restriction before the RNA transcription. Have you got any 
>advice what DNA purification method would yield pure enough DNA that I 
>would get rid of this problem? 
>
>So far I've tried 1) phenol-chloroform extraction without subsequent ether 
>treatment, 2) Geneclean II, 3) Qiagen's Qiaquick PCR purification. None 
>of these give me consistently pure DNA. They work sometimes for the RNA 
>transcription and sometimes they don't. 
>-- 
>Sami

I usually start with cesium banded plasmid (tip: during alkaline lysis, remove
RNA with LiCl rather than RNaseA), although Qiagen midis seem to work. I
linearize 5-10ug (overdigest, and avoid 3'overhangs), and clean up with
phenol alone (I used to add SDS/ProtK, which always worked, but proved to
be superfluous in my hands). After EtOH ppt, resuspend to ~200ng/ul. My
reaction conditions are:
        1x rxn buffer (40mMTris, pH7.9; 5mM MgCl2; 2mM spermidine)
        1mM rNTPs
        10mM DTT
        5u RNAsin (Promega)
        cap analog (if needed) to 600uM
        H2O (milli-Q)
        5-10u RNAP (T7 or SP6)
        0.2-1ug DNA (add last!)
1) assemble reactions at room temperature
2) cook at 37oC-42oC (for SP6) for 1-2hrs.
3) a trace amount of 3H-UTP is conveniently added to follow %incorporation
        and yield.
4) RNAs can be aliquoted and stored, or DNase-treated, phenol extracted,
        EtOH ppt'd, etc.
5) Typical yields are 30-90% incorporation (~0.4-1.18ug/ul of rxn).

Sami, you didn't mention anything about your electrophoresis setup, but there
could be RNases causing you problems there. A quick nondenaturing (to RNA)
agarose gel can be run in TAE/0.2% SDS (SDS in running buffer, not gel!),
which
can be helpful. Before staining with EtBr, remove SDS by soaking (20-30') in
H2O. Happy transcribing!


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




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