>>>>URGENT : IN VITRO TRANSCRIPTION <<<<<
jow at helix.nih.gov
Fri Oct 7 08:37:11 EST 1994
In article <belin-260994184027 at anapat1.univ-lyon1.fr> CJF 90/10 INSERM,
belin at cimacpcu.univ-lyon1.fr writes:
>What I need is to know if somebody has some experience on the subject and
>how to purify my mRNAs extracts after in-vitro transcription!
For what it's worth, I have had success using this method which I got
from Dirk Eick in Mu:nchen:
One hundred microliters (1x10^7) nuclei were thawed on ice (takes about
5-10 minutes) and were mixed with 100yl reaction buffer (10mM TrisHCl,
pH 8.0, 5mM MgCl2, 300mM KCl, 0.5mM each of ATP, CTP, GTP and 100yCi of
[alpha-32P]-UTP (800Ci/mmol, Amersham) and incubated for 20 minutes at
28!C. DNase I was added to a final concentration of 10yg/ml and the
incubation was continued for 5 minutes at 28!C. After the addition of
200ul STE buffer (100mM TrisHCL, pH7.5, 50mM EDTA, 0.5% SDS) and 20yl
proteinase K (10mg/ml, preincubated at 37! for 1h) the samples were
incubated for 1 h at 40!C. Nuclear transcripts were separated from
unincorporated nucleotides on a Sephadex G-50 (medium, DNA grade) column
(to just below the narrow waist, about 2ml, in a Pasteur pipet plugged
with a piece of glass fiber filter) equilibrated with 10mM TrisHCl, pH
7.5, 1mM EDTA, 1% SDS. Recovered labeled RNA in 0.6ml. The labelled RNA
was boiled for 10 minutes, chilled on ice and hybridized to DNA
oligonucleotides immobilized on a nylon membrane.
A similar method also worked for me. It added phenol extraction before
the G-50 column, and TCA precipitation, a short alkaline hydrolysis and
ethanol precipitation after the column. The alkaline hydrolysis is
important if any of the RNAs could hybridize to more than one of your
targets on the slot or dot blot.
I found the columns easier to work with when 2 to 3 mm of G-10 was
layered over the G-50. This retards formation of air channels in the
G-50. But Dirk doesn't do this.
Make sure that everything up to the hybridization is RNase-free.
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