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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