Bitan:Scintillation counting using the Triathler bench-top counter

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Scintillation counting of labeled RNA

Scintillation counting of labeled RNA

  1. After in vitro transcription and solubilization of RNA product in 100 μl (see In vitro transcription and RNA labeling) of the G-50 buffer, centrifuge the tube and keep 1 μl of RNA product in a 0.6-ml tube for scintillation counting.
  2. Perform counting using the Triathler bench-top scintillation counter (see below) and keep the aliquot for electrophoresis.
  3. Keep another 1-μl aliquot of the RNA product after G-50 purification and perform counting. Keep the aliquot for electrophoresis.
  4. To perform counting
    1. Start up the machine.
    2. 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”.
    3. If it does not read the latter, press “P-32” on the keyboard panel of the Triathler.
    4. 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.
    5. Insert the 0.6-ml tube with the 1-μl aliquot of RNA inside the plastic adaptor inside the counting chamber; close the lid.
    6. Press start.
    7. If counting many samples, remove the first tube, insert the second tube and press next.
    8. Jot down the counting values in cpm/μl.
    9. Divide the counting after G-50 purification to counting before purification to obtain percentage of incorporation of nucleotides (because G-50 removes unincorporated nucleotides).

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