Team:KAIST Korea/Notebook Labnote

From 2012.igem.org

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(Undo revision 94511 by Horang2k (talk))
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top:-70px;
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z-index: 2;
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#kaistcontent #inside
 +
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 +
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 +
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 +
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 +
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 +
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#kaistcontent #name
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#kaistcontent h3
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 +
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 +
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#kaistcontent ul
#kaistcontent ul
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 +
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 +
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 +
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 +
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 +
 +
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 +
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 +
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 +
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 +
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 +
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 +
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 +
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 +
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 +
 +
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 +
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 +
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 +
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 +
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 +
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 +
background-color: transparent;
 +
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 +
 +
.accordionContent #protocolcontent{
 +
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 +
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 +
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 +
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 +
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 +
font-family: 'Open Sans', sans-serif;
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 +
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 +
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 +
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 +
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 +
</head>
</head>
<body>
<body>
-
<div id="main">
+
 
-
<img id="top-img" src="https://static.igem.org/mediawiki/2012/4/4b/Top_image_team_kaist.png"/><!-- 이미지 넣어야대-->
+
<div id="main">
 +
<img id="top-img" src="https://static.igem.org/mediawiki/2012/6/6d/Back_home.PNG"/>
 +
 
 +
<div id="menucontent">
<div id="menucontent">
-
    <img src="https://static.igem.org/mediawiki/2012/4/4d/Note_ico.png"></img>
+
<img src="https://static.igem.org/mediawiki/2012/d/df/Part_ico.png"/></a>
-
        <a href="https://2012.igem.org/Team:KAIST_Korea/Notebook_Overview"><img src="https://static.igem.org/mediawiki/2012/e/e3/Overview_ico.png"/></a>
+
<a href="#"><img src="https://static.igem.org/mediawiki/2012/b/b0/Member_ico.png"/></a>
-
        <a href="https://2012.igem.org/Team:KAIST_Korea/Notebook_Protocol"><img src="https://static.igem.org/mediawiki/2012/b/b6/Protocol_ico.png"/></a>
+
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/c3/KAIST_ico.png"/></a>
-
        <a href="https://2012.igem.org/Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e0/Labnote_ico.png"/></a>
+
<a href="#"><img src="https://static.igem.org/mediawiki/2012/e/e6/Gallery_ico.png"/></a>
</div>
</div>
-
<div id="top-img-description-box"><span id="top-img-description">image description</span></div>
+
 
 +
<div id="top-img-description-box"><span id="top-img-description">aaaa</span></div>
 +
 
<img id="horizontal-line" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img>
<img id="horizontal-line" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img>
-
<div id="kaistcontent">
+
-
<div>
+
<div id="kaistcontent">
-
<img id="starter-grad" src="https://static.igem.org/mediawiki/2012/9/95/Starter_gradient_kaist.png"></img>
+
<ul class="tabs">  
-
        <h1>Labnote</h1>
+
        <li class="active" rel="tab1"> Call of Duty</li>
-
<span id="sub-title">Please enter sub title</span></br></br></br>
+
        <li rel="tab2"> Mortal Combat</li>
-
</br></br></br></br></br></br></br></br></br>
+
        <li rel="tab3"> Halo</li>
-
</div>
+
        <li rel="tab4"> Portal</li>
 +
    </ul>
 +
<div class="tab_container">  
 +
    <div id="tab1" class="tab_content">
 +
 +
       
 +
<div id="arc_wrapper">
 +
 +
 +
<div class="accordionButton"><div id="protocol">1. LB agar plate</div></div>
 +
<div class="accordionContent">
 +
<img style="margin:0 auto;float:none;"src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"></img>
 +
<div id="protocolcontent">
 +
<h3>Materials</h3>
 +
<img id="figure" alt="materials" src="https://static.igem.org/mediawiki/2012/4/46/KAIST_Protocol1_table1.png"></img>
 +
 +
<div style="clear:both;"></div>
 +
 +
</br></br>
 +
 +
<h3>Procedure</h3>
 +
<ol>
 +
<li>Add 25g LB broth and 15g agar into 1L DDW. </li>
 +
<li>Autoclave</li>
 +
</ol>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"></img>
 +
</div>
 +
 +
<div class="accordionButton"><div id="protocol">2. Genomic DNA Purification</div></div>
 +
<div class="accordionContent">
 +
<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
 +
<div id="protocolcontent">
 +
<h3>Materials</h3>
 +
 +
<img id="figure" style="width:632px" alt="materials" src="https://static.igem.org/mediawiki/2012/a/ac/Protocol2_table1.png"></img>
 +
<div style="clear:both;"></div>
 +
</br></br>
 +
 +
<h3>Procedure</h3></br>
 +
<h4>Pellet Cells</h4>
 +
<ol>
 +
<li>Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.</li>
 +
<li>Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.</li>
 +
<li>Remove the supernatant.</li>
 +
</ol>
 +
</br>
 +
<h4>Lyse Cells</h4>
 +
<ol start="4">
 +
<li>Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.</li>
 +
<li>Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.</li>
 +
<li>Remove the supernatant.</li>
 +
</ol>
 +
</br>
 +
<h4>Protein Precipitation</h4>
 +
<ol start="7">
 +
<li>Add 200ul of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20seconds to mix the Protein Precipitation Solution tieh the cell lysate.</li>
 +
<li>Incubate the sample on ice for 5minutes.</li>
 +
<li>Centrifuge at 13,000rpm 3minutes.</li>
 +
<li>Transfer the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube. </li>
 +
<span style="padding:none">Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.</span>
 +
<li>Add 600ul isopropanol.</li>
 +
<li>Gently mix by inversion until the thread-like strands of DNA form a visible mass.</li>
 +
<li>Centrifuge at 13000rpm for 2minutes.</li>
 +
<li>Carefully pour off the supernatant. </li>
 +
<li>Add 600ul of room temperature 70% ethanol and gently invert the tube 5 times to wash the DNA pellet. </li>
 +
<li>Centrifuge at 13000rpm for 2minutes. Carefully aspirate the ethanol. </li>
 +
<li>Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-14minutes. </li>
 +
<li>Add 50~100 μl TE buffer and rehydrate the DNA by incubating at 65℃ for 20min. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubationg the solution overnight at room temperature or at 4℃.</li>
 +
</ol>
 +
</br>
 +
<img id="figure" alt="TE buffer" src="https://static.igem.org/mediawiki/2012/d/df/KAIST_Protocol2_table2.png"></img>
 +
<div style="clear:both;"></div>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
 +
</div>
 +
 +
 +
<div class="accordionButton"><div id="protocol">3. Vector transformation</div></div>
 +
 +
<div class="accordionContent">
 +
<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
 +
<div id="protocolcontent">
 +
<h3>Procedure</h3></br>
 +
<h4>1) Restriction enzyme digestion</h4>
 +
<img id="figure" alt="Restriction enzyme digestion" src="https://static.igem.org/mediawiki/2012/2/27/Kaist_protocol3_table1.png"></img>
 +
<div style="clear:both;"></div>
 +
</br>
 +
<ol>
 +
<li>Incubate 2hr at 37℃.</li>
 +
<li>Inactivation 20min at 65℃.</li>
 +
</ol>
 +
</br>
 +
<h4>2) Dephosphorylation (Only for vector)</h4>
 +
<img id="figure" alt="Dephosphorylation (Only for vector)" src="https://static.igem.org/mediawiki/2012/2/2d/Kaist_protocol3_table2.png"></img>
 +
<div style="clear:both;"></div>
 +
</br>
 +
<ol>
 +
<li>Incubate 30min at 37℃.</li>
 +
<li>Inactivation 5min at 70℃.</li>
 +
</ol>
 +
</br>
 +
<h4>3) Ligation</h4>
 +
<img id="figure" alt="Ligation" src="https://static.igem.org/mediawiki/2012/3/31/Kaist_protocol3_table3.png"></img>
 +
<div style="clear:both;"></div>
 +
</br>
 +
<ol>
 +
<li>Incubate 16hr at 16℃.</li>
 +
</ol>
 +
</br>
 +
<h4>4) Transformation</h4>
 +
<ol>
 +
<li>Add 20ul ligated vector into 100ul of competent cell.</li>
 +
<li>Incubate ice 5min.</li>
 +
<li>Heat shock 42℃, 1min 30sec.</li>
 +
<li>Ice 5min.</li>
 +
<li>Recovery with 700ul LB at 37℃, 1hr.</li>
 +
<li>Plating.</li>
 +
</ol>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
 +
</div>
 +
 +
<div class="accordionButton"><div id="protocol">4. PCR</div></div>
 +
<div class="accordionContent">
 +
<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
 +
<div id="protocolcontent">
 +
<h3>Procedure</h3>
 +
<img id="figure" alt="PCR" src="https://static.igem.org/mediawiki/2012/0/02/Kaist_protocol4_table1.png"></img>
 +
</br></br>
 +
<h3>Reaction Conditions</h3>
 +
<ol>
 +
<li>95℃ 3min</li>
 +
<li>95℃ 30sec</li>
 +
<li>54℃ 30sec</li>
 +
<li>72℃ 1min/kbp</li>
 +
<li>72℃ 10min</li>
 +
<li>12℃ ∞</li>
 +
</ol>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
 +
</div>
 +
 +
<div class="accordionButton"><div id="protocol">5. Gel extraction</div></div>
 +
<div class="accordionContent">
 +
<img src="https://static.igem.org/mediawiki/2012/7/77/KAIST_Protocol_content_top.png" id="top-part"/>
 +
<div id="protocolcontent">
 +
<h3>Materials</h3>
 +
<ul>
 +
<li>MGTM Gel Extraction SV - Macrogen</li>
 +
</ul>
 +
</br>
 +
<h3>Procedure</h3>
 +
<ol>
 +
<li>Excise the DNA band of interest using an ethanol-cleaned razor blade.</li>
 +
<li>Weigh the gel slice in a microcentrifuge tube. Add 3 volumes (ul) of Buffer GB to 1 volume (mg) of gel.</li>
 +
<li>Incubate at 50°C until the agarose gel is completely melted (5 ~ 10 min).</li>
 +
<li>Transfer the mixture to a MGTM SV column. Centrifuge for 1 min. Discard the pass-through and re-insert the MGTM SV column into the Collection Tube.</li>
 +
<li>Add 700 ul of Buffer NW to the MGTM SV column. Centrifuge for 30 sec. Discard the pass-through. Reinsert the MGTM SV column into the Collection Tube.</li>
 +
<li>Centrifuge for additional 1 min to remove residual wash buffer. Transfer the MGTM SV column to a new 1.5 ml tube.</li>
 +
<li>Apply 30ul of Buffer EB to the center of the membrane in the MGTM SV column, let stand for 1 min, and centrifuge for 1 min.</li>
 +
</ol>
 +
</div>
 +
<img src="https://static.igem.org/mediawiki/2012/8/85/Protocol_content_bottom.png" id="bot-part"/>
 +
</div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
    </div><!-- #tab1 -->
 +
    <div id="tab2" class="tab_content">
 +
 +
      <p><img src="images/mortal combat.jpg"> <br />
 +
      <strong>
 +
      Mortal Kombat returns after a lengthy hiatus and puts players
 +
      back into the Tournament for 2D fighting with gruesome combat.
 +
      </strong>
 +
      </p>
 +
 +
    </div><!-- #tab2 -->
 +
    <div id="tab3" class="tab_content">
 +
 +
      <p><img src="images/halo.jpg"> <br />
 +
      <strong>
 +
      Halo: Reach is the culmination of the superlative combat, sensational
 +
      multiplayer, and seamless online integration that are the hallmarks
 +
      of this superb series.
 +
      </strong>
 +
      </p>
 +
 +
    </div><!-- #tab3 -->
 +
    <div id="tab4" class="tab_content">
 +
 +
      <p><img src="images/portal.jpg"> <br />
 +
      <strong>
 +
      Portal 2 is an action shooter game from Valve Software that draws
 +
      from the original formula of innovative gameplay, story, and music,
 +
      which earned the original Portal more than 70 industry accolades.
 +
      </strong>
 +
      </p>
 +
 +
    </div><!-- #tab4 -->
 +
   
 +
</div> <!-- .tab_container -->
 +
 +
 +
 +
 +
 +
 +
</br>
 +
</br>
 +
</div>
</div>
</div>
</div>
 +
<script type="text/javascript">
 +
 +
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 +
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 +
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 +
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 +
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</body>
</body>
</html>
</html>
{{:Team:KAIST_Korea/footer}}
{{:Team:KAIST_Korea/footer}}

Revision as of 10:12, 4 September 2012

KAIST Korea 2012 iGEM

aaaa
  • Call of Duty
  • Mortal Combat
  • Halo
  • Portal
1. LB agar plate

Materials

materials


Procedure

  1. Add 25g LB broth and 15g agar into 1L DDW.
  2. Autoclave
2. Genomic DNA Purification

Materials

materials


Procedure


Pellet Cells

  1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
  2. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
  3. Remove the supernatant.

Lyse Cells

  1. Add 1ml of an overnight culture to a 1.5ml microcentrifuge tube.
  2. Centrifuge at 13000 rpm for 2.5minutes to pellet the cells.
  3. Remove the supernatant.

Protein Precipitation

  1. Add 200ul of Protein Precipitation Solution to the RNase-treated cell lysate. Vortex vigorously at high speed for 20seconds to mix the Protein Precipitation Solution tieh the cell lysate.
  2. Incubate the sample on ice for 5minutes.
  3. Centrifuge at 13,000rpm 3minutes.
  4. Transfer the supernatant containing the DNA to a clean 1.5ml microcentrifuge tube.
    5. Note: Some supernatant may remain in the original tube containing the protein pellet. Leave this residual liquid in the tube to avoid contaminating the DNA solution with the precipitated protein.
  5. Add 600ul isopropanol.
  6. Gently mix by inversion until the thread-like strands of DNA form a visible mass.
  7. Centrifuge at 13000rpm for 2minutes.
  8. Carefully pour off the supernatant.
  9. Add 600ul of room temperature 70% ethanol and gently invert the tube 5 times to wash the DNA pellet.
  10. Centrifuge at 13000rpm for 2minutes. Carefully aspirate the ethanol.
  11. Drain the tube on clean absorbent paper and allow the pellet to air-dry for 10-14minutes.
  12. Add 50~100 μl TE buffer and rehydrate the DNA by incubating at 65℃ for 20min. Periodically mix the solution by gently tapping the tube. Alternatively, rehydrate the DNA by incubationg the solution overnight at room temperature or at 4℃.

TE buffer
3. Vector transformation

Procedure


1) Restriction enzyme digestion

Restriction enzyme digestion

  1. Incubate 2hr at 37℃.
  2. Inactivation 20min at 65℃.

2) Dephosphorylation (Only for vector)

Dephosphorylation (Only for vector)

  1. Incubate 30min at 37℃.
  2. Inactivation 5min at 70℃.

3) Ligation

Ligation

  1. Incubate 16hr at 16℃.

4) Transformation

  1. Add 20ul ligated vector into 100ul of competent cell.
  2. Incubate ice 5min.
  3. Heat shock 42℃, 1min 30sec.
  4. Ice 5min.
  5. Recovery with 700ul LB at 37℃, 1hr.
  6. Plating.
4. PCR

Procedure

PCR

Reaction Conditions

  1. 95℃ 3min
  2. 95℃ 30sec
  3. 54℃ 30sec
  4. 72℃ 1min/kbp
  5. 72℃ 10min
  6. 12℃ ∞
5. Gel extraction

Materials

  • MGTM Gel Extraction SV - Macrogen

Procedure

  1. Excise the DNA band of interest using an ethanol-cleaned razor blade.
  2. Weigh the gel slice in a microcentrifuge tube. Add 3 volumes (ul) of Buffer GB to 1 volume (mg) of gel.
  3. Incubate at 50°C until the agarose gel is completely melted (5 ~ 10 min).
  4. Transfer the mixture to a MGTM SV column. Centrifuge for 1 min. Discard the pass-through and re-insert the MGTM SV column into the Collection Tube.
  5. Add 700 ul of Buffer NW to the MGTM SV column. Centrifuge for 30 sec. Discard the pass-through. Reinsert the MGTM SV column into the Collection Tube.
  6. Centrifuge for additional 1 min to remove residual wash buffer. Transfer the MGTM SV column to a new 1.5 ml tube.
  7. Apply 30ul of Buffer EB to the center of the membrane in the MGTM SV column, let stand for 1 min, and centrifuge for 1 min.


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