Bitan:Scintillation counting using the Triathler bench-top counter
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border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'> <tr> <td width=443 valign=top style='width:442.8pt;border:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt'> <p class=MsoNormal align=center style='text-align:center'><span style='font-family:Calibri'>Scintillation counting of labeled RNA<o:p></o:p></span></p> <ol style='margin-top:0cm' start=1 type=1> <li class=MsoNormal style='mso-list:l2 level1 lfo2;tab-stops:list 36.0pt'><span style='font-family:Calibri'>After </span><span style='font-family:Calibri; mso-fareast-language:KO'><i>in vitro</i></span><span style='font-size: 16.0pt;font-family:Calibri'> </span><span style='font-family:Calibri'>transcription and solubilization of RNA product in 100 μl (see <i><a href="http://openwetware.org/wiki/Bitan:In-vitro_transcription%2C_labeling_and_G-50_purification_of_RNA">In vitro<span style='font-style:normal'> transcription and RNA labeling</span></a></i></span><span style='font-family:Calibri'>) 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></span></li>
<li class=MsoNormal style='mso-list:l2 level1 lfo2;tab-stops:list 36.0pt'><span style='font-family:Calibri'>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></span></li> <li class=MsoNormal style='mso-list:l2 level1 lfo2;tab-stops:list 36.0pt'><span style='font-family:Calibri'>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></span></li> <li class=MsoNormal style='mso-list:l2 level1 lfo2;tab-stops:list 36.0pt'><span style='font-family:Calibri'>To perform counting <o:p></o:p></span></li> <ol style='margin-top:0cm' start=1 type=a> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>Start up the machine.<o:p></o:p></span></li>
<li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>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></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>If it does not read the latter, press “P-32” on the keyboard panel of the Triathler.<o:p></o:p></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>Insert the provided plastic scintillation adaptor into the counting chamber. This allows measurement of low-volume, high-energy, dry counting of the <sup>32</sup>P-labeled RNA samples. This also precludes the need to mix the sample with the scintillation fluid.<o:p></o:p></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>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></span></li>
<li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>Press start.<o:p></o:p></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>If counting many samples, remove the first tube, insert the second tube and press next.<o:p></o:p></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>Jot down the counting values in cpm/μl.<o:p></o:p></span></li> <li class=MsoNormal style='mso-list:l2 level2 lfo2;tab-stops:list 72.0pt'><span style='font-family:Calibri'>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></span></li> </ol> </ol>
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