Problems 5' labelling of total RNA
nospam_jweiner1 at ix.urz.uni-heidelberg.de
Thu Jun 22 11:14:06 EST 2000
I have enormous problems while 5' labelling of total RNA with
polynucleotide kinase and gamma-32P-ATP. 10ug of total RNA is treated with
2U of shrimp alkaline phosphatase prior to the kinase reaction, then
phenol/chlorophorm extracted. After SAP treatment it is still OK and
undigested. About 5-10 ug of SAP treated RNA is incubated for 40' with 20U
of PNK and 50 uCi of gamma-32P-ATP in 20ul with 40 U of RNAse Inhibitor.
RNA visualized on a radioactive PAGE immediatly after this reaction seems
to be RNAse digested: quite clear bands are visible on the film much below
the expected smallest RNA band visible on EtBr stained PAGEs of normal,
untreated total RNA, and there is only very little radioactivity where the
bulk of the tRNAs and rRNAs should be expected... any ideas? Maybe the SAP
treatment is not sufficient and the broken RNAs are preferentially
labelled? What do you think?
"A moose once bit my sister.."
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