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phosphatase removal in RNA samples

phosphatase removal in RNA samples - Protocols and Methods Forum

phosphatase removal in RNA samples - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 10-23-2008, 02:53 PM
Simone Marker
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Default phosphatase removal in RNA samples



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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Old 10-23-2008, 08:05 PM
WS
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Default phosphatase removal in RNA samples

On Oct 23, 4:53*pm, "Simone Marker" <[Only registered users see links. ]-kl.de> wrote:
Hi Simone,

two things you could try:

1) add a tiny little bit of phenol to the sample. This will kill the
phosphatease. No need for any extraction.
2) If this should not be an option, you might add 100mM sodium
fluoride to the gel buffers to inhibit the phosphatase. Will increase
conductivity however and thus heat production during electrophoresis.

Best reagrds,

Wo,
from Bochum


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