Team:UC Davis/Notebook/Protocols

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   <div id="newnavi">
   <div id="newnavi">
     <ul class="newmenu">
     <ul class="newmenu">
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     <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
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     <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
           <ul>
           <ul>
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           <li><a href="https://2012.igem.org/">Main iGEM</a></li>
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           <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
           <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
           <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
           <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
           <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
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             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
title="Data">Ethylene Glycol</a></li>
title="Data">Ethylene Glycol</a></li>
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<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
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title="Data">Modeling</a></li>
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             <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
           </ul>
           </ul>
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<a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery"><img src="https://static.igem.org/mediawiki/2012/4/43/UCD_Gallery_small_banner-1.jpg"></a>
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     </div>
     </div>
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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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<p>Procedure</p>
<p>Procedure</p>
<ul>
<ul>
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<li>1. Prepare a starter culture of MG1655 <i>E. coli</i> with pBad regulated and his-tagged LC-Cutinase along with a negative control.</li>
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<li>Prepare a starter culture of MG1655 <i>E. coli</i> with pBad regulated and his-tagged LC-Cutinase along with a negative control.</li>
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<li>2. Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li>
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<li>Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li>
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<li>3. Let the cultures grow until and OD 600 of 0.5, then inoculate two 100 mL cultures (one for the his-tagged protein and one for the negative control). </li>
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<li>Let the cultures grow until and OD 600 of 0.5, then inoculate two 100 mL cultures (one for the his-tagged protein and one for the negative control). </li>
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<li>4. Once these cultures are at an OD of 0.8, separate into two 50 mL cultures. Induce one at 10 uM, leave the other uninduced.</li>
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<li>Once these cultures are at an OD of 0.8, separate into two 50 mL cultures. Induce one at 10 uM, leave the other uninduced.</li>
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<li>5. Take 1 mL samples at different time points.</li>
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<li>Take 1 mL samples at different time points.</li>
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<li>6. Centrifuge samples at 5000g for 5 minutes then take off and save media.</li>
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<li>Centrifuge samples at 5000g for 5 minutes then take off and save media.</li>
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<li>7. Wash cells with water.</li>
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<li>Wash cells with water.</li>
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<li>8. Resuspend with 300 uL of B-PER Protein Extraction Reagent and follow the instructions provided with that kit. After this, there should be samples of culture media, and soluble and insoluble whole cell lysate.</li>
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<li>Resuspend with 300 uL of B-PER Protein Extraction Reagent and follow the instructions provided with that kit. After this, there should be samples of culture media, and soluble and insoluble whole cell lysate.</li>
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<li>9. Take 15 uL of each sample, including controls, and run on western blot.</li>
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<li> Take 15 uL of each sample, including controls, and run on western blot.</li>
     </div>
     </div>
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     </div>
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<p class="menu_head">SDM (Site Directed Mutagenesis)</p>
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    <div class="menu_body">
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 +
<p>Materials</p>
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<ul>
 +
<li>Pfu turbo</li>
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<li>10X Pfu turbo buffer</li>
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<li>dNTPs (10mM)</li>
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<li>Forward and reverse primers (0.1ug/uL, see methods section for design tips)</li>
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<li>dH<sub>2</sub>0</li>
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<li>Dpn1</li>
 +
<li>Competent cells</li>
 +
</ul>
 +
<br>
 +
<p>Methods</p>
 +
<ul>
 +
<li>Primer Design</li>
 +
<ul>
 +
<li>Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this.</li>
 +
<li>Primers should have a minimum GC content of 40% and terminate in one or more C's or G's.</li>
 +
<li>Tm should be greater or equal to 78°C and can be calculated as follows:</li>
 +
<li>Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch</li>
 +
</ul></ul>
 +
<ul>
 +
<li>Reaction</li>
 +
<ul>
 +
<li>Template DNA: 1 µL</li>
 +
<li>10X Buffer: 5 µL</li>
 +
<li>Forward Primer (100 ng/µL): 1µL</li>
 +
<li>Reverse Primer (100 ng/µL): 1µL</li>
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<li>10mM dNTPs: 1µL</li>
 +
<li>Pfu Turbo (Stratagene): 1µL</li>
 +
<li>MilliQ H<sub>2</sub>0: 40µL</li>
 +
</ul>
 +
<li>PCR Program</li>
 +
<ul>
 +
<li>95°C for 1 minute</li>
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<li>95°C for 50 seconds, 60°C for 50 seconds, 68°C for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total</li>
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<li>68°C for 7 minutes</li>
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<li>4°C hold</li>
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<li>Following PCR - save 4µL of PCR reaction.</li>
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<li>To remaining 46µLs add 1µL of Dpn1 to PCR reaction (PCR cleanup is not necessary). Incubate at 37°C for 1-2 hours to digest parental DNA. Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern. Transform into competent cells (PCR cleanup is not necessary).</li>
 +
</ul>
 +
 +
<br>
 +
<li>Protocol Adapted From <a href="http://mcmanuslab.ucsf.edu/protocol/site-directed-mutagenesis-stratagene-protocol">Here</a>
 +
</ul>
 +
    </div>
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 +
 +
</div>
</div>
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href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
Ethylene Glycol</a> </li><li><a style="color:#000000 "
Ethylene Glycol</a> </li><li><a style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis/Data/Modeling ">
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Modeling</a> </li><li><a style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>
href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>

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