Team:UC Davis/Notebook/Protocols

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<p class="menu_head">Construction of a Transposase Illumina Sequencing Library</p>
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    <div class="menu_body">
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<p>Procedure</p>
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<ul>
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<li>Extract genomic DNA using any proprietary gDNA extraction kit. Once DNA is extracted, quantify your DNA and run it on an agarose gel to check DNA quality.</li>
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<li>Prepare Illumina Adapters for Transposase Priming, Mix the Following:
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      <ul><li>10µM No_PCR_Adapter_1: 10µL</li>
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      <li>10µM No_PCR_Adapter_2: 10µL</li>
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      <li>10µM Mosaic Element: 20µL</li>
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      <li>5M NaCl: 1.2µL</li>
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      <li>Incubate at 98C for 5 mins and let cool to room temperature</li></ul></li>
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<li>Prime Transposase
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      <ul><li>Transposase: 10µL</li>
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      <li>Annealed adapter (above): 5µL</li>
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      <li>Water: 15µL</li>
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      <li>Incubate at room temperature for 20-30 mins.</li></ul></li>
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<li>Tagmentation Reaction
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      <ul><li>Primed Transposase: 10µL (depends on the concentration of available Tn5)</li>
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      <li>200ng of gDNA extration: XµL</li>
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      <li>5x Tn5 Buffer: 3µL</li>
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      <li>Water: up to 15µL</li>
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      <li>Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform</li>
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      <li>Incubate at 55C for 10 mins, and immediately pool reactions and clean up using a PCR clean up kit, elute with 15µL water</li></ul></li>
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<li>Gap Filling
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      <ul><li>Run a typical PCR reaction, without oligos, at 72C for 5 mins</li></ul></li>
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<li>Size Selection
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      <ul><li>Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform</li></ul></li>
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<li>Quality check your library
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      <ul><li>Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.</li></ul></li>
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<li>SUBMIT!</li>
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</ul>
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</div>
<p class="menu_head">Cutinase Expression and Western Blot</p>
<p class="menu_head">Cutinase Expression and Western Blot</p>

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