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RNA cleavage assay with Fluorescece?

rna cy5 In vitro Cleavage RNase

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#1 gabri

gabri

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Posted 05 November 2012 - 01:38 AM

Hello Everybody!

I would like to perform RNA cleavage assay avoiding the usage of radioactivity.
I would like to mark my RNA with fluorescence (Cy5) in 5´. THen I mix it with my RNase and then I will run it on a poliacrillamide gel. For fluorescence scan I plan to use the Odysee analyser from licor.
Since I am not familiar with fluorescent labelling, I have some questions:
- How much RNA do I need to load on a gel so that the fluorescent is strong enough to be detected?
- How do I label my RNA? I was thinking to order an oligo RNA already labelled in 5´ and then ligate it with my RNA with t4-RNA ligase. Is this the right way?

I thank for the help!

Gabriella





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