Labeling large RNA's

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Labeling large RNA's

Postby nekameneka » May 14 2017 11:51 pm

Hello everyone, I have a question about labeling RNA. Not decided which label, probably biotin because right now I have a kit but my worry is that I have quiet large RNA and using calculation they provide in the protocol I came to a silly number. They say I need 0.6nmol of 5' ends and that should correspond to 100ug of 500bp double stranded nucleic acid or I can use this equation to calculate: A/BxCx1000 nmols/ul where A is concentration of nucleic acid, B is average MW of nucleotide (317ug/umol for RNA) and C is total number of base pairs.
Let's say when I purify my RNA I have a sample with usually around 300ng/ul. If I use the formula to calculate how much of the sample I need I come to the result that I need 8ml of this sample to be able to do the reaction properly. Impossible! Can someone help me with getting this right?
Thanks a lot!
nekameneka
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