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Scintillation counting of labeled RNA<o:p></o:p>
- After in vitro transcription
and solubilization of RNA product in 100 μl (see <a
href="http://openwetware.org/wiki/Bitan:In-vitro_transcription%2C_labeling_and_G-50_purification_of_RNA">In
vitro transcription and RNA labeling</a>) of the G-50 buffer, centrifuge the tube
and keep 1 μl of RNA product in a 0.6-ml tube for scintillation
counting. <o:p></o:p>
- Perform counting using the Triathler
bench-top scintillation counter (see below) and keep the aliquot for <a
href="http://openwetware.org/index.php?title=Bitan:TBE-urea-polyacrylamide_gel_electrophoresis&action=edit&redlink=1">electrophoresis</a>.<o:p></o:p>
- Keep another 1-μl aliquot of the RNA
product after G-50 purification and perform counting. Keep the aliquot
for <a
href="http://openwetware.org/index.php?title=Bitan:TBE-urea-polyacrylamide_gel_electrophoresis&action=edit&redlink=1">electrophoresis</a>.<o:p></o:p>
- To perform counting <o:p></o:p>
- Start up the machine.<o:p></o:p>
- Machine reads “Clear Saved Parameters”,
“Powering up .. Version 1.8” and then either “<H-3> Ready start
sys edit” or “P-32 Ready start sys edit”. <o:p></o:p>
- If it does not read the latter, press
“P-32” on the keyboard panel of the Triathler.<o:p></o:p>
- Insert the provided plastic scintillation
adaptor into the counting chamber. This allows measurement of low-volume,
high-energy, dry counting of the 32P-labeled RNA samples.
This also precludes the need to mix the sample with the scintillation
fluid.<o:p></o:p>
- Insert the 0.6-ml tube with the 1-μl
aliquot of RNA inside the plastic adaptor inside the counting chamber;
close the lid.<o:p></o:p>
- Press start.<o:p></o:p>
- If counting many samples, remove the first
tube, insert the second tube and press next.<o:p></o:p>
- Jot down the counting values in
cpm/μl.<o:p></o:p>
- Divide the counting after G-50 purification
to counting before purification to obtain percentage of incorporation
of nucleotides (because G-50 removes unincorporated nucleotides).<o:p></o:p>
<a
href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a><o:p></o:p>
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