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Increase in internal control band over titration

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#1 DNAlooper



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Posted 24 July 2014 - 09:13 PM

I have a peculiar problem while performing in vitro transcription assay. I am testing a promoter X and its repression by transcriptional repressor Y. For this I have cloned the promoter X fragment containing 89 bp of downstream transcribing region into a plasmid containing transcriptional terminator. Repressor Y binds to upstream promoter region which I have included in the construct. I also have an internal control - RNA 1 within this template plasmid. For invitro transcription assay, when I add the repressor protein with increasing concentrations, and constant RNA polymerase (E.coli) I see the decrease in the 89bp mRNA band which is expected. (See the attached image. Upper band is promoter mRNA, lower band is RNA 1 control) But I also see increase in the internal control band intensity (RNA 1) which is totally unexpected !! The internal control RNA band is supposed to be constant. Some words of mouth said that it is due to increase in RNA polymerase load on the RNA 1 since the promoter X mRNA is repressed by the repressor Y. Can anyone please explain why this happens? 


Attached Thumbnails

  • RNA 1 load.png

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