Bitan:In-vitro transcription, labeling and G-50 purification of RNA

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In vitro transcription and RNA labeling<o:p></o:p>

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For large-scale transcription, RiboMAXTM 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>.  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>

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T7 Transcription 5X Buffer                                                                 20 μl<o:p></o:p>

100 mM rATP                                                                                      7.5 μl<o:p></o:p>

100 mM rUTP                                                                                     7.5 μl<o:p></o:p>

100 mM rGTP                                                                                     7.5 μl<o:p></o:p>

100 mM rCTP                                                                                      1 μl<o:p></o:p>

α-32P-rCTP (3,000 Ci/mmol, 10 μCi/μl)                                              2 μl if fresh, otherwise if 2 half-lives have passed use 3–5 μl<o:p></o:p>

Purified template DNA (5–10 μg) in nuclease-free water               44.5 μl (5–10 μg)<o:p></o:p>

Enzyme Mix (T7)                                                                                 10 μl   <o:p></o:p>

TOTAL                                                                                     100 μl<o:p></o:p>

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Removal of template DNA<o:p></o:p>

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Use manufacturer’s procedure in the RiboMAXTM Large Scale RNA Production Systems–T7 kit. <o:p></o:p>

  1. 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.  (e.g. when adding 5 μg of template DNA, use 10–20 μl DNase.).  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>
  2. 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>).  Mix by a vortex for 1 min (mixture turns cloudy/milky), and centrifuge at 16,000 g for 2 min.  Transfer the upper (aqueous) phase to a new tube or aspirate and discard the lower phase.  Take proper precaution in handling radioactive solid and liquid waste.  <o:p></o:p>
  3. Add 1 volume (100 μl) of chloroform:isoamyl alcohol (24:1, <a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=C0549%7CSIGMA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPEC">#C0549, Sigma</a>).  Mix by a vortex for 1 min and centrifuge at 16,000 g for 2 min.  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>
  4. 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.  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>
  5. Mix by vortex and place in the freezer for 30 min.  <o:p></o:p>
  6. To precipitate RNA, spin at 16,000 g or top speed for 30 min in the temperature-controlled centrifuge at 4 °C.<o:p></o:p>
  7. Carefully aspirate the supernate and wash the pellet with 0.8 μl 70% ethanol.  Dry the pellet at 37 °C using a heat block. <o:p></o:p>
  8. 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>.  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>

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Removal of unincorporated nucleotides<o:p></o:p>

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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">#27-5330-01, GE Healthcare</a>) according to the manufacturer’s procedure.  Each column can be used for up to 50 μl solution to be desalted.  <o:p></o:p>

  1. Pre-spin the column at 3,000 rpm (740 g) for 1 min, and collect the buffer in the collection tube provided.  <o:p></o:p>
  2. Add the sample to be desalted onto the centre of the G-50 resin within the tube without perturbing the resin with the tip.  Avoid applying the sample to the sides of the tube.<o:p></o:p>
  3. 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 g) to collect desalted RNA.<o:p></o:p>
  4. 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>

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