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></li>
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    <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
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          <ul>
 +
          <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
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          <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
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          <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
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         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
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         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
           <ul>
           <ul>
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             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a></li>
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             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
 +
title="Data">Ethylene Glycol</a></li>
 +
<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
 +
title="Data">Modeling</a></li>
 +
 
             <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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<ul>
<ul>
<li>EMS (Sigma)</li>
<li>EMS (Sigma)</li>
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<li>50mL Conical Corning tubes</li>
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<li>50mL conical Corning tubes</li>
<li>15mL Falcon tubes</li>
<li>15mL Falcon tubes</li>
<li>LB Broth</li>
<li>LB Broth</li>
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<p>Procedure</p>
<p>Procedure</p>
<ul>
<ul>
-
<li>1. Prepare a 10mL liquid culture in LB in a 50mL Conical tube and grow it overnight until it reaches OD 0.2.</li>
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<li>1. Prepare a 10mL liquid culture in LB in a 50mL conical tube and grow it overnight until it reaches OD 0.2.</li>
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
<li>Wash twice with 10mL Buffer A.</li>
<li>Wash twice with 10mL Buffer A.</li>
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     </div>
     </div>
 +
<p class="menu_head">Construction of a Transposase Illumina Sequencing Library</p>
 +
    <div class="menu_body">
 +
 +
<p>Procedure</p>
 +
<ul>
 +
<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>
 +
<li>Prepare Illumina Adapters for Transposase Priming, Mix the Following:
 +
      <ul><li>10µM No_PCR_Adapter_1: 10µL</li>
 +
      <li>10µM No_PCR_Adapter_2: 10µL</li>
 +
      <li>10µM Mosaic Element: 20µL</li>
 +
      <li>5M NaCl: 1.2µL</li>
 +
      <li>Incubate at 98C for 5 mins and let cool to room temperature</li></ul></li>
 +
<li>Prime Transposase
 +
      <ul><li>Transposase: 10µL</li>
 +
      <li>Annealed adapter (above): 5µL</li>
 +
      <li>Water: 15µL</li>
 +
      <li>Incubate at room temperature for 20-30 mins.</li></ul></li>
 +
<li>Tagmentation Reaction
 +
      <ul><li>Primed Transposase: 10µL (depends on the concentration of available Tn5)</li>
 +
      <li>200ng of gDNA extration: XµL</li>
 +
      <li>5x Tn5 Buffer: 3µL</li>
 +
      <li>Water: up to 15µL</li>
 +
      <li>Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform</li>
 +
      <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>
 +
<li>Gap Filling
 +
      <ul><li>Run a typical PCR reaction, without oligos, at 72C for 5 mins</li></ul></li>
 +
<li>Size Selection
 +
      <ul><li>Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform</li></ul></li>
 +
<li>Quality check your library
 +
      <ul><li>Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.</li></ul></li>
 +
<li>SUBMIT!</li>
 +
</ul>
</div>
</div>
 +
 +
<p class="menu_head">Cutinase Expression and Western Blot</p>
 +
    <div class="menu_body">
 +
 +
<p>Procedure</p>
 +
<ul>
 +
<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>
 +
<li>Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li>
 +
<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>
 +
<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>
 +
<li>Take 1 mL samples at different time points.</li>
 +
<li>Centrifuge samples at 5000g for 5 minutes then take off and save media.</li>
 +
<li>Wash cells with water.</li>
 +
<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>
 +
<li> Take 15 uL of each sample, including controls, and run on western blot.</li>
 +
 +
    </div>
 +
 +
<p class="menu_head">pNPB Assay</p>
 +
    <div class="menu_body">
 +
 +
<p>Materials</p>
 +
<ul>
 +
<li>pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity)
 +
<ul><li>10mM pNPB in acetonitrile</li>
 +
<li>1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)</li></ul></li>
 +
<li>LB with specific resistance</li>
 +
<li>Cell Culture</li>
 +
<li>96 well plate</li></ul>
 +
<br>
 +
<p>Protocol</p>
 +
<ul><li>First assign each well of the plate except for outer wells which should be filled with LB to prevent evaporation of medium.</li>
 +
<li>Fill wells with 95 uL of LB</li>
 +
<li>Fill wells with 5 uL of cell culture</li>
 +
<li>In fumehood, add 100 uL of bufer to each well</li>
 +
<li>Run in Tecan taking both ODs and absorbance 405 readings</li>
 +
 +
    </div>
 +
 +
<p class="menu_head">SDM (Site Directed Mutagenesis)</p>
 +
    <div class="menu_body">
 +
 +
<p>Materials</p>
 +
<ul>
 +
<li>Pfu turbo</li>
 +
<li>10X Pfu turbo buffer</li>
 +
<li>dNTPs (10mM)</li>
 +
<li>Forward and reverse primers (0.1ug/uL, see methods section for design tips)</li>
 +
<li>dH<sub>2</sub>0</li>
 +
<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>
 +
<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>
 +
<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>
 +
<li>68°C for 7 minutes</li>
 +
<li>4°C hold</li>
 +
<li>Following PCR - save 4µL of PCR reaction.</li>
 +
<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>
 +
 +
 +
 +
 +
</div>
 +
 +
 +
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<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"  >
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<ul style="font-size:10px;list-style-image:none;list-style-type:none;float:left;display:inline;color:#000000;"
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  >
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<li style="float:left;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul style="text-indent:-15px;
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<li style="float:left;margin:0 10px;"><a
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list-style-image:none;list-style-type:none;color:#000000;"><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis">Welcome</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis">Tweets</a></li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis">Sponsors</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a> </li> </ul> </a> </li>
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href="https://2012.igem.org/Team:UC_Davis"><p>Home</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
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style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis">Welcome</a> </li><li><a
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style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis">Tweets</a></li><li><a
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style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis">Sponsors</a> </li><li><a
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style="color:#000000 "
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href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a> </li>
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</ul> </a> </li>
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<li style="float:left;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p><ul style="text-indent:-15px;
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<li style="float:left;margin:0 10px;"><a
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list-style-image:none;list-style-type:none;color:#000000;"><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Team">Who we are</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Team">Students</a></li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Team">Advisors</a> </li> </ul> </a> </li>
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href="https://2012.igem.org/Team:UC_Davis/Team"><p>Team</p><ul
 +
style="text-indent:-15px;
 +
list-style-image:none;list-style-type:none;color:#000000;"><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Who we are</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Students</a></li><li><a
 +
style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Team">Advisors</a> </li>
 +
</ul> </a> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a> <ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module Engineering</a></li>
+
<li style="float:left ;margin:0 10px;"><a
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering ">Protein Engineering</a></li>
+
href="https://2012.igem.org/Team:UC_Davis/Project "><p>Project</p></a>
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis Engineering</a></li>
+
<ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000"><li><a
-
   <li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution "> - Directed Evolution</a></li>
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style="color:#000000 "
-
   <li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> - Rational Engineering</a></li>
+
href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a>
-
<li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li></ul> </li>
+
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst ">Module
 +
Engineering</a></li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering
 +
">Protein Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Strain ">Chassis
 +
Engineering</a></li>
 +
   <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution ">
 +
- Directed Evolution</a></li>
 +
   <li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain "> -
 +
Rational Engineering</a></li>
 +
<li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a>
 +
</li></ul> </li>
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a> <a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
+
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Safety "> <p>Safety</p></a>
 +
<a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Safety "> Safety</a> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Notebook "> <p>Notebook</p></a> <ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000  "><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li> </ul> </li>
+
<li style="float:left ;margin:0 10px;"><a
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook ">
 +
<p>Notebook</p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
  "><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols
 +
">Protocols</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria">Critera</a> </li>
 +
</ul> </li>
-
<li style="float:left ;margin:0 10px;"><a href="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000  "><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Data "> Data</a> </li><li><a style="color:#000000 " href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>
+
<li style="float:left ;margin:0 10px;"><a
 +
title="https://2012.igem.org/Team:UC_Davis/Data "> <p>Data </p></a> <ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
  "><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity ">
 +
Cutinase Activity</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol ">
 +
Ethylene Glycol</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Data/Modeling ">
 +
Modeling</a> </li><li><a style="color:#000000 "
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Team:UC_Davis/Attributions "> <p>Attribution </p></a><a style="color:#000000 "  href="https://2012.igem.org/Team:UC_Davis/Attributions "> Attribution</a></li>
+
href="https://2012.igem.org/Team:UC_Davis/Parts ">Parts</a></li> </ul>
-
<li style="float:left ;margin:0 10px"><a href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><a style="color:#000000 " href="https://2012.igem.org/Main_Page " > iGEM</a></li>
+
<li style="float:left ;margin:0 10px"><a
-
</ul>
+
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
<p>Attribution </p></a><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Attributions ">
 +
Attribution</a></li>
 +
 
 +
<li style="float:left ;margin:0 10px"><a
 +
href="https://2012.igem.org/Main_Page "> <p>iGEM </p></a><ul
 +
style="text-indent:-15px;list-style-image:none;list-style-type:none;color:#000000
 +
"><li><a style="color:#000000 " href="https://2012.igem.org/Main_Page
 +
">Main iGEM</a> </li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Criteria "> Criteria</a>
 +
</li><li><a style="color:#000000 "
 +
href="https://2012.igem.org/Team:UC_Davis/Human_Practices ">Human
 +
Practices</a></li> </ul>
</div>
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