in vitro transcription

Darren Boehning boehnin1 at jeflin.tju.edu
Thu Jan 29 17:04:31 EST 1998


In article <3.0.32.19980121141145.009223c0 at facstaff.wisc.edu>, 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

I have done ALOT of in vitro transcription, and this is generally caused by 
either RNAse contamination in the reaction (as stated), or is caused many 
times by RNases in the agarose!  Use either a formamide gel (which I hate), or 
"pre-run" your gel with no sample in it for about a half hour before you load 
your samples.  Tubes are usually the culprit in the rxn, and I find if I use a 
commercially available RNAse decontaminate (such as RNASE AWAY, this is not a 
product endorsement) the problem is resolved.  Also, this may be a stupid 
point, but make sure you are using an RNA quench buffer and are heating the 
samples to 70-80C for 10min before loading.



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