Bitan:In-vitro transcription and RNA labeling

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<div class=Section1>

<table border=1 cellspacing=0 cellpadding=0 style='border-collapse:collapse;

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;mso-fareast-language:KO'><b><i><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></i></b></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri;mso-fareast-language:KO'><b><i>In vitro</i></b></span><span
 style='font-family:Calibri'><b> transcription and RNA labeling<o:p></o:p></b></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri'><b><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>Fo</span><span
 style='font-family:Calibri;mso-fareast-language:JA'>r large-scale
 transcription, </span><span style='font-family:Calibri'>RiboMAX<sup>TM</sup>
 Large Scale RNA Production Systems-T7 (<a
 href="http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_39&amp;ckt=1">#P1300,
 Promega</a>) was used according to <a
 href="http://www.promega.com/tbs/tb166/tb166.html">manufacture’s protocol</a>.<span
 style="mso-spacerun: yes">&nbsp; </span>Thaw reagents at room temperature and
 mix the transcription buffer and the rNTPs by a vortex. Make sure to combine
 the reaction components at room temperature to avoid precipitation of DNA or
 dNTPs in the presence of spermidine when cold. Spermidine is a component of
 the transcription buffer. After mixing, incubate at 37 °C for 4 h or
 overnight.<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>T7 Transcription 5X
 Buffer<span style='mso-tab-count:6'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>20
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>100 mM rATP<span
 style='mso-tab-count:8'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>7.5
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>100 mM rUTP<span
 style='mso-tab-count:8'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>7.5
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>100 mM rGTP<span
 style='mso-tab-count:8'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>7.5
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>100 mM rCTP<span
 style='mso-tab-count:8'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>1
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-left:324.0pt;text-indent:-324.0pt'><span
 style='font-family:Calibri'>&#945;</span><span style='font-family:Calibri;
 mso-fareast-language:JA'>-</span><span style='font-family:Calibri'><sup>32</sup>P-rCTP
 (3,000 Ci/mmol, 10 &#956;Ci/&#956;l)<span style='mso-tab-count:1'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>2
 &#956;l if fresh, otherwise if 2 half-lives has passed use 3&#8211;5 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>Purified template DNA
 (5&#8211;10 mg) in nuclease-free water<span style='mso-tab-count:2'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>44.5
 &#956;l (5&#8211;10 &#956;g)<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'><u>Enzyme Mix (T7)<span
 style='mso-tab-count:7'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>10
 &#956;l</u><span style="mso-spacerun: yes">&nbsp;&nbsp; </span><u><o:p></o:p></u></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>TOTAL<span
 style='mso-tab-count:8'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span>100
 &#956;l<o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri'><b>Removal of template DNA<o:p></o:p></b></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri'><b><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>Use manufacturer’s
 procedure in the RiboMAX<sup>TM</sup> Large Scale RNA Production
 Systems&#8211;T7 kit. <o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=1 type=1>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Add RQ1 RNase-Free DNase (1 U/&#956;l) to a
      concentration of 1 U/&#956;g of the template DNA used; incubate at 37 °C
      for 15 min.<span style="mso-spacerun: yes">&nbsp; </span>(<i>e.g.</i></span><span
      style='font-family:Calibri'> when adding 5 &#956;g of template DNA, use
      10&#8211;20 &#956;l DNase.).<span style="mso-spacerun: yes">&nbsp;
      </span>It is found that this duration as recommended by the manufacturer
      is not sufficient to degrade completely the template DNA used. Increase
      this incubation reaction to 3&#8211;4 hours if residual DNA is detected
      in the negative control used for RT-PCR (to be linked). This can cause
      background amplification during SELEX and may render cycles futile
      because of the persistence of initial library sequences.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Extract with 1 volume (100 &#956;l) of
      acetate-saturated phenol (pH 4):chloroform:isoamyl alcohol mixture
      (125:24:1, <a
      href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=ja&amp;N4=77619%7CFLUKA&amp;N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&amp;F=SPEC">#77619,
      Sigma</a>).<span style="mso-spacerun: yes">&nbsp; </span>Mix by a vortex
      for 1 min (mixture turns cloudy/milky), and centrifuge at 16,000 <i>g</i></span><span
      style='font-family:Calibri'> for 2 min.<span style="mso-spacerun:
      yes">&nbsp; </span>Transfer the upper (aqueous) phase to a new tube or
      aspirate and discard the lower phase.<span style="mso-spacerun:
      yes">&nbsp; </span>Take proper precaution in handling radioactive solid
      and liquid waste.<span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Add 1 volume (100 &#956;l) of
      chloroform:isoamyl alcohol (24:1, <span style='color:black'><a
      href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=C0549%7CSIGMA&amp;N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&amp;F=SPEC">#C0549,
      Sigma</a></span>).<span style="mso-spacerun: yes">&nbsp; </span>Mix by a
      vortex for 1 min and centrifuge at 16,000 <i>g</i></span><span
      style='font-family:Calibri'> for 2 min.<span style="mso-spacerun:
      yes">&nbsp; </span>Transfer the upper (aqueous) layer to a new tube or
      aspirate and discard the bottom phase. If residual phenol remains,
      repeat centrifugation and aspirate from the bottom phase.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Add 0.1 volume (10 &#956;l) of 3M sodium
      acetate (pH 5.2) provided and 1 volume (100 &#956;l) of isopropanol or
      2.5 volumes (250 &#956;l) of 100% ethanol.<span style="mso-spacerun:
      yes">&nbsp; </span>Isopropanol is advantageous because it keeps the
      unincorporated rNTPs in solution and the volume used is less than
      ethanol (useful with larger volumes of phenol-extracted RNA, to be
      linked to RNA elution)<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Mix by vortex and place in the freezer for
      30 min.<span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>To precipitate RNA, spin at 16,000 <i>g </i></span><span
      style='font-family:Calibri'>or top speed for 30 min in the
      temperature-controlled centrifuge at 4 °C.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Carefully aspirate the supernate and wash
      the pellet with 0.8 &#956;l 70% ethanol.<span style="mso-spacerun:
      yes">&nbsp; </span>Dry the pellet at 37 °C using a heat block. <o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l4 level1 lfo2;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Suspend RNA in 1 volume (100 &#956;l) of
      nuclease-free water or the riboprobe buffer that comes in Illustra
      Microspin <a
      href="http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&amp;productId=9987477&amp;catalogId=29104&amp;matchedCatNo=45001398%7C%7C45001399&amp;pos=8&amp;catCode=RE_SC&amp;endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&amp;fromCat=yes&amp;keepSessionSearchOutPut=true&amp;fromSearch=Y&amp;searchKey=50%7C%7CG&amp;highlightProductsItemsFlag=Y">G-50
      kits</a>.<span style="mso-spacerun: yes">&nbsp; </span>Store at
      &#8722;70 °C for long-term storage, or &#8211;20 °C for next-day usage,
      or follow on to the next step.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal><span style='font-family:Calibri'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri'><b>Removal of unincorporated nucleotides<o:p></o:p></b></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'><![if !supportEmptyParas]>&nbsp;<![endif]><o:p></o:p></span></p>
 <p class=MsoNormal><span style='font-family:Calibri'>Use MicroSpin G-50 spin
 columns (<a
 href="http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&amp;productId=9987477&amp;catalogId=29104&amp;matchedCatNo=45001398%7C%7C45001399&amp;pos=8&amp;catCode=RE_SC&amp;endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&amp;fromCat=yes&amp;keepSessionSearchOutPut=true&amp;fromSearch=Y&amp;searchKey=50%7C%7CG&amp;highlightProductsItemsFlag=Y">#<span
 style='mso-fareast-language:JA'>27-5330-01, </span>GE Healthcare</a>)
 according to the manufacturer’s procedure.<span style="mso-spacerun:
 yes">&nbsp; </span>Each column can be used for up to 50 &#956;l solution to
 be desalted.<span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></p>
 <ol style='margin-top:0cm' start=1 type=1>
  <li class=MsoNormal style='mso-list:l0 level1 lfo5;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Pre-spin the column at 3,000 rpm (740 <i>g</i></span><span
      style='font-family:Calibri'>) for 1 min, and collect the buffer in the
      collection tube provided.<span style="mso-spacerun: yes">&nbsp; </span><o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l0 level1 lfo5;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Add the sample to be desalted onto the
      centre of the G-50 resin within the tube without perturbing the resin
      with the tip.<span style="mso-spacerun: yes">&nbsp; </span>Avoid
      applying the sample to the sides of the tube.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l0 level1 lfo5;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Put the column assembly into a clean
      Eppendorf (<a href="file://localhost/Products.php">O-ring capped</a>).
      This time spin for 2 min at 3,000 rpm (740 <i>g</i></span><span
      style='font-family:Calibri'>) to collect desalted RNA.<o:p></o:p></span></li>
  <li class=MsoNormal style='mso-list:l0 level1 lfo5;tab-stops:list 36.0pt'><span
      style='font-family:Calibri'>Store at &#8722;70 °C for long-term storage,
      or &#8211;20 °C for next-day usage, or follow on to the next step.<o:p></o:p></span></li>
 </ol>
 <p class=MsoNormal align=center style='text-align:center'><span
 style='font-family:Calibri'><a href="http://openwetware.org/wiki/Bitan:todo">Back
 to To-Do List</a></span><o:p></o:p></p>
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