RNase protection assay and radiolysis
pamela.norton at mail.tju.edu
Wed Jan 2 15:28:53 EST 2002
In article <184.108.40.206.2.20011226115626.009ef200 at mail.albany.edu>, James
A. Dutko <jd4300 at albany.edu> wrote:
> Thanks in advance for any insight and advice.
> I am performing RNase protection assay with gel purified in vitro
> synthesized transcripts. I label the riboprobes with UTP to specific
> activities of 1-2 X 10e8 per microgram - the message is rare. One probe is
> 500 nt (protects 465 nt) and the other is 350 nt (protects 258 nt) long
> each with approx 25% U's. I store the probe in 0.5M NH4OAc, 0.2% SDS, and
> 1mM EDTA at -80C. After about 3 or 4 days the probe no longer detects the
> messages. Could this be due primarily to radiolysis of the probe? Could I
> extend the life of my probes by changing the storage conditions? If
> radiolysis is the nature of the beast for riboprobes then I can set up the
> experiments differently. Thanks again.
I think that radiolysis likely is significant. What fraction of the U's
do you expect will be labelled?
You can determine the amount of intact probe by running a small amount
on a gel after 3-4 days - how does it look? If you really need probes
of high specific activity, you might need to re-synthesize for each
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