phosphatase removal in RNA samples

Simone Marker via methods%40net.bio.net (by marker from rhrk.uni-kl.de)
Thu Oct 23 09:53:36 EST 2008


Hi,

I am looking for chemical properties of short RNA species (siRNA) and I want 
to know wheather they are 5' phosphorylated (e.g. mono-or triphosphate). I 
want to compare their migration behaviour in a denaturing polyacrylamide gel 
(7M urea), which is also influenced by the number of phosphates. I also need 
some RNA samples that are completely dephosphorylated, to compare the 
migration.
So I would use calf intestinal alkaline phosphatase (better than SAP with 
RNA), but I do not want to remove the phosphatase by phenol-chlorophorm 
extraction (to much loss of RNA, not quatitative). Do you think that removal 
is necessary? I just want to load the treated samples on the gel (near the 
untreated ones) and see their migration behaviour (in northern). Does active 
phosphatase influence the migration of the RNA in denaturing gels? (since 
CIP is not heat-inactivatable).

Thank you for your help,
Simone 




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