Team:UC Davis/Protocols

From 2013.igem.org

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     </div>
     </div>
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<p class="menu_head">Sequencing Preparation (Quintara Biosciences)</p>
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<p class="menu_head">Sequencing Preparation</p>
     <div class="menu_body">
     <div class="menu_body">
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<p>Procedure</p>
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<p>Procedure (Quintara Biosciences)</p>
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 µM.</li>
<li>Primer will have [ng] content printed on label: add 10x H<sub>2</sub>0 for DNA at 100 µM.</li>
<li>Need 10 µM solution for sequencing, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL H<sub>2</sub>0).</li>
<li>Need 10 µM solution for sequencing, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL H<sub>2</sub>0).</li>
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         <p>Procedure</p>
         <p>Procedure</p>
<ol>
<ol>
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<li>Grow cultures overnight in LB at 37 C, 150 RPM. </li>
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<li>Grow cultures overnight, or until OD<sub>600</sub>>1, in LB at 37 C, 150 RPM. </li>
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li>
<li>Measure OD<sub>600</sub> and dilute to get <0.01 OD<sub>600</sub>.</li>
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li>
<li>Grow until the OD<sub>600</sub> approaches 0.5.</li>
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<li>Load 96 well plate with LB or M9, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. </li>
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<li>Load 96 well plate with LB, or other media of choice, depending on the experiment, as well as the appropriate antibiotic, inducer stock solutions, and the appropriate volume of culture so as to reach an OD<sub>600</sub> of 0.1 in 200 µL. If changing media, first spin down the culture to pellet, decant and aspirate any remaining media, and resuspend in new media to reach the desired OD<sub>600</sub> before loading in plate.</li>
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<li>Be sure to include the appropriate positive and negative controls.</li>
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<li>Be sure to include the appropriate positive and negative controls (e.g. blank media, strain control, etc.).</li>
</ol>
</ol>
<table class="gray">
<table class="gray">
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<li> Identify site that needs to be mutated.</li>
<li> Identify site that needs to be mutated.</li>
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li>
<li>Check the amino acid sequence to create a silent mutation, generally the last base in a codon.</li>
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<li>Check a codon usage table to help choose how the codon should be changed, try to pick a frequently used codon. </li>
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<li>Check a codon usage table for your chassis to help choose how the codon should be changed, try to pick a frequently used codon. </li>
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<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer, try to have it start and end in a G or C. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center.  </li>
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<li>Take about 20 base pairs upstream and 20 base pairs downstream of your desired mutation site to create your primer. The sequence should be identical to the template except for the one changed base you are trying to mutate at the center.  </li>
<li>The reverse primer will be the reverse complement of this sequence.</li>
<li>The reverse primer will be the reverse complement of this sequence.</li>
</ol>
</ol>
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     <div class="menu_body">
     <div class="menu_body">
                 <p>Materials</p>
                 <p>Materials</p>
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<li>Primer will have [µg] content printed on label: add H<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li>
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<li>Primer will have [µg] content printed on label: add dH<sub>2</sub>0 1:1 for DNA at 1 µg/µL.</li>
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<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x.</li>
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<li>Need 0.1 µg/µL for PCR reaction, so dilute a portion of the hydrated primer solution 10x (e.g. 1 µL primer + 9 µL dH<sub>2</sub>0).</li>
<li>Determine DNA concentration of template DNA.</li>
<li>Determine DNA concentration of template DNA.</li>
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<li>1 µL      dNTPs (10 mM)</li>
<li>1 µL      dNTPs (10 mM)</li>
<li>1 µL      Pfu Turbo (enzyme)</li>
<li>1 µL      Pfu Turbo (enzyme)</li>
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<li>Add appropriate amount of dH<sub>2</sub>O.</li>
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<li>Add appropriate amount of dH<sub>2</sub>O to reach total volume.</li>
50 µL Total
50 µL Total
<br></br>
<br></br>

Latest revision as of 22:26, 19 October 2013

Protocols