synthetic RNA stability/degradation

[NICHOLAS THEODORAKIS]

In article <D0tq9E.2vo at ucc.su.oz.au>,
robynb <robynb at westmed.wh.su.edu.au> wrote:
>I have a problem with synthetic RNA seemingly breaking down upon
>storage.
>
>I have been using any techniques I know of to keep the RNA free from
>RNases, such as nuclease-free water (Promega), filter pipette tips,
>nuclease-free tubes -which I do not treat in any other way-, addition
>of guanidium thiocyanate to the end stage of transcription and rRNasin
>is added to the RT mix.
>
>However, when I store the RNA in working aliquots, at -70oC, the first
>couple of times I do an RT-PCR (using a fresh aliquot each time) it
>seems to work fine, but in subsequent RT-PCRs I seem to lose the PCR
>product.
>
>Does anyone have a clue about what could be happening? I would
>appreciate any suggestions at all. Please contact me through my email
>address or via this news group.
>
>Robyn Biti
>Dept. Immunology, Westmead Hospital. Australia.
>ROBYNB at westmed.wh.su.edu.au

This was happening to a friend of mine.  He changed several things in his 
protocol, but what seemed to make the most sense was that he thought he 
might have residual RNase in his plasmid DNA template (he used RNAse in 
his plasmid preps).  When he treated his template with Proteinase K, his 
problem was solved.

						Nick


-- 
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
			Nick Theodorakis
			ntheo at welchlink.welch.jhu.edu
			Johns Hopkins Medical School, Baltimore, MD