Team:Calgary/Notebook/Protocols/Process3

From 2011.igem.org

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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>
TITLE=cDNA Synthesis/Reverse Transcription Reaction|
TITLE=cDNA Synthesis/Reverse Transcription Reaction|
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<li>Thaw all components (except enzyme), mix thoroughly and centrifuge before use.</li>
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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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<div style="margin-bottom:60px">
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  <table width="700">
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    <tr>
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      <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="reaction conditions"></a> </p></div></td>
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      <td><b>5 min at 25°C</b></td>
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    <tr>
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      <td>30 min at 42°C</td>
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    </tr>
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    <tr>
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      <td>5 min at 85°C</td>
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      <td>variable</td>
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    <tr>
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      <td>Hold at 4°C</td>
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</table>
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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>

Revision as of 17:08, 27 September 2011

{{Team:Calgary/Notebookbar|


  • Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li>
  • Add the following to a 0.2mL thin-walled PCR tube sitting on ice:</li> TITLE=cDNA Synthesis/Reverse Transcription Reaction| BODY=

    cDNA Synthesis by Reverse Transcription

    Component Volume
    RNA (40ng – 10ng to 1μg total RNA/rxn) variable
    Nuclease free water variable
    qScript cDNA synthesis mix 4 uL
    Final Volume 20 uL
  • Thaw all components (except enzyme), mix thoroughly and centrifuge before use.
  • Mix components by gently vortexing and then centrifuge 4s to collect contents.
  • Incubate in a PCR machine using the following conditions:

    		•
    5 min at 25°C
    30 min at 42°C
    5 min at 85°C variable
    Hold at 4°C
  • 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).
    • Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process3"