Team:Calgary/Notebook/Protocols/Process3

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<p>The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, and is used to generate cDNA from isolate RNA (from <i>Pseudomonas spp.</i>) using reverse transcriptase.</p>
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<ol>
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<li>Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li>
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<li>Add the following to a 0.2mL thin-walled PCR tube sitting on ice:</li>
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       <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td>
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       <td><b>Component</b></td>
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      <td><b>Volume</b></td>
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    </tr>
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      <td>RNA (40ng – 10ng to 1μg total RNA)</td>
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      <td>variable</td>
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      <td>Nuclease free water</td>
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      <td>variable</td>
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      <td>qScript cDNA synthesis mix</td>
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      <td>4 μL</td>
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      <td>Final Volume</td>
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      <td>20 μL</td>
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<li>Mix components by gently vortexing and then centrifuge 4s to collect contents.</li>
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<li>Incubate in a PCR machine using the following conditions:</li>
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       <td><b>Reagent</b></td>
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       <td><CENTER>5 min at 25°C</CENTER></td>
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      <td><b>Volume ( 1x )</b></td>
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      <td><b>Volume ( 3x )</b></td>
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      <td><b>Volume ( 5x )</b></td>
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      <td><b>Volume ( 15x )</b></td>
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       <td>Sterile H2O</td>
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       <td><CENTER>30 min at 42°C</CENTER></td>
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      <td>36 μL</td>
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      <td>108 μL</td>
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      <td>180 μL</td>
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      <td>540 μL</td>
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      <td><CENTER>5 min at 85°C</CENTER></td>
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      <td><CENTER>Hold at 4°C</CENTER></td>
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<li>After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range.  Do 3 replicates of each PCR reaction.  Run PCRs on a real-time PCR machine (e.g. ABI 7900).</li>
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Latest revision as of 02:59, 29 September 2011


cDNA Synthesis/Reverse Transcription Reaction

The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, and is used to generate cDNA from isolate RNA (from Pseudomonas spp.) using reverse transcriptase.

  1. Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.
  2. Add the following to a 0.2mL thin-walled PCR tube sitting on ice:
    3. Component Volume
    RNA (40ng – 10ng to 1μg total RNA) variable
    Nuclease free water variable
    qScript cDNA synthesis mix 4 μL
    Final Volume 20 μL
  3. Mix components by gently vortexing and then centrifuge 4s to collect contents.
  4. Incubate in a PCR machine using the following conditions:
    5. 5 min at 25°C
    30 min at 42°C
    5 min at 85°C
    Hold at 4°C
  5. After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range. Do 3 replicates of each PCR reaction. Run PCRs on a real-time PCR machine (e.g. ABI 7900).






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