in vitro transcription

brett brett at BORCIM.WUSTL.EDU
Thu Jan 22 12:41:48 EST 1998


>On Thu, 22 Jan 1998 13:09:33 +0000, Warren Hirst <w.hirst at ic.ac.uk>
>wrote:
>
>>Sounds like you have RNase contamination in those samples that give a 
>>smear after electrophoresis. Make sure that you are working in RNase 
>>free conditions. Use a new bag of 1.5ml eppendorf tubes (from a good 
>>supplier, these are normally RNase free), same with tips etc. Try 
>>including some recombinant RNasin (Promega), if you haven't already. The 
>>fact that you are seeing degradation with some samples and not others 
>>could suggest that the cDNA template could be contaminated. Make sure 
>>that the template is clean. Re-extract with phenol/chloroform if 
>>neccesary. Good luck.
>>
>>
>>ryyukhan at FACSTAFF.WISC.EDU wrote:
>>> 
>>> We are doing a lot of transcription usingSP6 and T7 from Promega following
>>>their recomedations.
>>> with some templateswe have a satisfactory results ( not very good) but
>>>sometimes the labeling just go bad ( just smear after electrophoresis).
>>> any suggestion what can be done.
>>> Thank you
>>> 
>>> Rus

>What warren has described is one explanation for your smear; another
>would be incomplete linearization of your template (if your are using
>plasmids as templates) , in this case make sure that the restriction
>digest is complete(by agarose elpho).

This is not likely to cause this. Incomplete linearization will lead to
"run-around" transcripts of large size that: i) will consume your reagents;
and ii) won't appear as a smear, rather as a large single band (high MW -
not resolved).

>Another explanation is that the polymerase drops off the template to
>early, thus causing shorter transcripts due to a too high reaction
>temperature.

Also not likely - SP6 and T7 are highly processive. I prefer to do my SP6
reactions at 42C and have never seen this.

My guess is RNase contamination.  Make up all new reagents if they are
suspect. Clean up templates using ProtK/SDS, followed by 2 rounds of phenol
extraction.  Use RNasin/DTT in your reactions.  The other thing I have seen
in a student I taught transcription to was smearing possibly due to incomplete
removal of organic solvents from the template. Also, eschew all "Mongo
Transcription" kits - their idea of RNase-free means you don't have to pay
for it.  Finally, read up on the reaction.  I think there is a nice treatment
of the subject in Meth Enzym V180 by Yisraeli and Melton.
Brett Lindenbach
brett at borcim.wustl.edu

Dept Molecular Microbiology
Washington University School of Medicine
Box 8230
660 S. Euclid Ave
St Louis, MO 63110




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