phosphatase removal in RNA samples

[Barbara MacGregor]

Hi Simone,

Antarctic phosphatase (New England Biolabs) is heat-inactivatible,  
that might work.

Barbara MacGregor

>
>
> From: "Simone Marker" <marker from rhrk.uni-kl.de>
> Date: October 23, 2008 10:53:36 AM EDT
> To: methods from net.bio.net
> Subject: phosphatase removal in RNA samples
> Reply-To: Simone Marker <marker from rhrk.uni-kl.de>
>
>
> 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
>
>