Lisa J Brunet
brunet at uclink2.berkeley.edu
Wed Aug 24 12:33:55 EST 1994
J.P. Hays (jph5 at leicester.ac.uk) wrote:
: Dear Colleagues,
: I am having great difficulty in maintaining stocks of "viable",
: (i.e RT-PCRable), viral mRNA, and would be very grateful of any suggestions.
: Initially, I extract viral positive sense RNA using RNAzol, (Biogenesis), and
: store the product in 50 microlitres TE buffer with 0.25 microlitres RNasin,
: (Promega). The components for the TE buffer are individually produced as stock
: solutions, autoclaved, and mixed when required. The RNA is stored in DEPC
: treated, sterilised, Eppendorfs, (though the glassware in which the TE buffer
: is stored and the water used in the TE buffer components are not DEPC treated).
: RT-PCR on freshly isolated RNA works well, but after 2 days at -20oC
: the RNA seems to have disappeared!
: Thanks for any advice!
: John Hays
: jph5 at le.ac.uk
RNasin requires DTT to remain active. Otherwise it degrades releasing
RNase. You may try adding DTT to your RNA stocks or leaving out the
RNasin altogether. I just store my RNA in TE at -80 with no problems.
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