Bitan:In-vitro transcription, labeling and G-50 purification of RNA
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<o:DocumentProperties> <o:Author>Farid Rahimi</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Farid Rahimi</o:LastAuthor> <o:Revision>4</o:Revision> <o:Created>2009-12-03T00:43:00Z</o:Created> <o:LastSaved>2009-12-03T00:48:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Words>988</o:Words> <o:Characters>3855</o:Characters> <o:Company>UCLA</o:Company> <o:Lines>257</o:Lines> <o:Paragraphs>257</o:Paragraphs> <o:CharactersWithSpaces>6919</o:CharactersWithSpaces> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
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<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]> <![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]> <![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&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"> </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]> <![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'> </span>20 μl<o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri'>100 mM rATP<span style='mso-tab-count:8'> </span>7.5 μl<o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri'>100 mM rUTP<span style='mso-tab-count:8'> </span>7.5 μl<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-family:Calibri'>100 mM rGTP<span style='mso-tab-count:8'> </span>7.5 μl<o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri'>100 mM rCTP<span style='mso-tab-count:8'> </span>1 μl<o:p></o:p></span></p> <p class=MsoNormal style='margin-left:324.0pt;text-indent:-324.0pt'><span style='font-family:Calibri'>α</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 μCi/μl)<span style='mso-tab-count:1'> </span>2 μl if fresh, otherwise if 2 half-lives have passed use 3–5 μl<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-family:Calibri'>Purified template DNA (5–10 μg) in nuclease-free water<span style='mso-tab-count:2'> </span>44.5 μl (5–10 μg)<o:p></o:p></span></p> <p class=MsoNormal><span style='font-family:Calibri'><u>Enzyme Mix (T7)<span style='mso-tab-count:7'> </span>10 μl</u><span style="mso-spacerun: yes"> </span><u><o:p></o:p></u></span></p> <p class=MsoNormal><span style='font-family:Calibri'>TOTAL<span style='mso-tab-count:8'> </span>100 μl<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-family:Calibri'><![if !supportEmptyParas]> <![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]> <![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–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/μl) to a
concentration of 1 U/μg of the template DNA used; incubate at 37 °C
for 15 min.<span style="mso-spacerun: yes"> </span>(<i>e.g.</i></span><span
style='font-family:Calibri'> when adding 5 μg of template DNA, use
10–20 μl DNase.).<span style="mso-spacerun: yes">
</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 3–4 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 μl) of
acetate-saturated phenol (pH 4):chloroform:isoamyl alcohol mixture
(125:24:1, <a
href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=ja&N4=77619%7CFLUKA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">#77619,
Sigma</a>).<span style="mso-spacerun: yes"> </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"> </span>Transfer the upper (aqueous) phase to a new tube or
aspirate and discard the lower phase.<span style="mso-spacerun:
yes"> </span>Take proper precaution in handling radioactive solid
and liquid waste.<span style="mso-spacerun: yes"> </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 μl) of
chloroform:isoamyl alcohol (24:1, <span style='color:black'><a
href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=C0549%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">#C0549,
Sigma</a></span>).<span style="mso-spacerun: yes"> </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"> </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 μl) of 3M sodium
acetate (pH 5.2) provided and 1 volume (100 μl) of isopropanol or
2.5 volumes (250 μl) of 100% ethanol.<span style="mso-spacerun:
yes"> </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"> </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 μl 70% ethanol.<span style="mso-spacerun:
yes"> </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 μl) 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&productId=9987477&catalogId=29104&matchedCatNo=45001398%7C%7C45001399&pos=8&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=50%7C%7CG&highlightProductsItemsFlag=Y">G-50
kits</a>.<span style="mso-spacerun: yes"> </span>Store at
−70 °C for long-term storage, or –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]> <![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]> <![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&productId=9987477&catalogId=29104&matchedCatNo=45001398%7C%7C45001399&pos=8&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=50%7C%7CG&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"> </span>Each column can be used for up to 50 μl solution to be desalted.<span style="mso-spacerun: yes"> </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"> </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"> </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 −70 °C for long-term storage,
or –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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