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Ilian Atanassov, 02/19/2015 11:48 AM

1 1 Ilian Atanassov 
h1. Digestion protocol
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* Starting amounts 1 and 10 micrograms of RNA (both N2 and P6661 and P6662 worms)





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* Enzymes:





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> # Snake Venom Phosphodiesterase - to cleave RNA - http://www.sigmaaldrich.com/catalog/product/sigma/p3243?lang=de&region=DE





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> # Bacterial Phosphatase - to dephopshorylate nucleotides to nucleosides - http://www.carlroth.com/catalogue/catalogue.do?favOid=0000000b000320fd00010023&act=showBookmark&lang=en-nl&market=NL





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* Digestion conditions 





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> Original - 50mM Tris (pH 7.5 - 9) 





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> Preferred due to LC-MS/MS compatibility - 0.1 M ammonium bicarbonate, pH 8.0 or 0.1M TEAB pH 8





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* Sample precipitation for LC-MS/MS analysis





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> Ethanol precipitation using 1/10 sample volume 3M sodium acetate pH 5.3 and 3 times sample volume absolute EtOH (-20 deg). Incubation for >2h to overnight. Centrifugation at 13000 (4 deg) and dry supernatant for analysis.





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This step precipitates the digestion enzymes and undigested material. Dephosphorylated nucleosides remain in the supernatant.