RNase protection probe activities

Dan Pereira mgs6145 at student.health.ufl.edu
Thu Aug 10 10:58:02 EST 1995


macdiarm at sbsu1.auckland.ac.nz (Colin MacD) wrote:
>
> 
> 
> -- 
> I am setting up an RNAse protection assay and have been having problems with degradation (i.e. radiolytic decay) of my 32P-labelled probes. My probes have specific activities of about 5x10^9 cpm/microgram. I dont get degradation using low sp. activity probes, but need high sensitivity.
> Is 5x10^9 too hot?
> What is the best probe size and activity for high sensitivity?
> I would greatly appreciate any help.
> 
> Dave Whittaker
> whittakr at sbsu1.auckland.ac.nz

I think 10^9 is a bit high.  I typically use isotope that is at a specific
activity of 800 Ci/mmol for synthesis of the probe.  I then use 1-2 x10^5 cpms
of probe/reaction.  My detection sensitivity is quite good and as long as you
use the probe right away, degradation should be minimal.  Best of Luck.



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