phosphatase removal in RNA samples

[WS]

On Oct 23, 4:53 pm, "Simone Marker" <mar... from rhrk.uni-kl.de> wrote:
> 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
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

> 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