Team:Chiba/Notebook 1

From 2010.igem.org

(Difference between revisions)
 
(25 intermediate revisions not shown)
Line 1: Line 1:
-
<!--- Background Contents --->
 
<html>
<html>
-
<body bgcolor="white" text="black" link="yellow" vlink="purple" alink="red" background="https://static.igem.org/mediawiki/2010/a/a1/Chiba_ground_2.jpg">
+
<style type="text/css">
-
<img src="https://static.igem.org/mediawiki/2010/2/2a/Chiba_home2.jpg" width="965" height="230">
+
#top-section {
 +
    height: 0px;
 +
    margin-top: -80px;
 +
    margin-left: auto;
 +
    margin-right: auto;
 +
    margin-bottom: 0 !important;
 +
    margin-bottom: 0px;
 +
    padding:0;
 +
    border: 0;
 +
    }
 +
</style>
 +
</html>
 +
<!--- Background Contents --->
 +
<!--- 1st tab --->
 +
<html><br><br><br>
 +
<div id="search-controls">
 +
<form action="/Special:Search" id="searchform">
 +
<input id="searchInput" name="search" type="text" title="Search 2010.igem.org [f]" accesskey="f" value="" />
 +
<input type='submit' name="go" class="searchButton" id="searchGoButton" value="Go" title="Go to a page with this exact name if exists" />&nbsp;
 +
      <input type='submit' name="fulltext" class="searchButton" id="mw-searchButton" value="Search" title="Search the pages for this text" />
 +
</form>
 +
</div> <!-- close search controls -->
 +
 
 +
 
 +
<body bgcolor="blue" text="black" link="yellow" vlink="purple" alink="red" background="https://static.igem.org/mediawiki/2010/a/a1/Chiba_ground_2.jpg">
 +
<img src="https://static.igem.org/mediawiki/2010/f/f9/Chiba_menu_2.jpg">
<p>
<p>
<style type="text/css">  
<style type="text/css">  
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
-
        <html xmlns="http://www.w3.org/1999/xhtml">
+
<html xmlns="http://www.w3.org/1999/xhtml">
-
                <head>
+
        <head>
-
                        <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
+
                <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
-
                        <title>Free CSS Navigation Menu Designs 1 at exploding-boy.com</title>
+
                <title>Free CSS Navigation Menu Designs 2 at exploding-boy.com</title>
-
                        <style type="text/css">
+
                <style type="text/css">
<!--
<!--
     body {
     body {
Line 25: Line 49:
}
}
-
/*- Menu Tabs E--------------------------- */
+
img {
 +
border: none;
 +
}
-
     #tabsE {
+
/*- Menu Tabs 10--------------------------- */
 +
 
 +
     #tabs10 {
       float:left;
       float:left;
       width:100%;
       width:100%;
-
      background:#000;
 
       font-size:100%;
       font-size:100%;
       line-height:normal;
       line-height:normal;
-
 
+
      background:#373737;
 +
      margin-top:-10px;
       }
       }
-
     #tabsE ul {
+
     #tabs10 ul {
-
        margin:0;
+
          margin:0;
-
        padding:2px 10px 0 50px;
+
          padding:2px 10px 0 105px;
-
        list-style:none;
+
          list-style:none;
       }
       }
-
     #tabsE li {
+
     #tabs10 li {
       float:left;
       float:left;
       display:inline;
       display:inline;
 +
      margin:0;
       padding:0;
       padding:0;
-
       margin-right: 40px;
+
       margin-left:40px;
-
 
+
       }
       }
-
     #tabsE li ul {
+
     #tabs10 a {
-
position: absolute;
+
-
left: -999em;
+
-
    }
+
-
   
+
-
    #tabsE li:hover ul {
+
-
      float:left;
+
-
      color: white !important;
+
-
+
-
    }
+
-
+
-
    #tabsE li:hover ul, #tabsE li.sfhover ul {
+
-
      left:auto;
+
-
      padding: 10px;
+
-
      color: white !important;
+
-
    }
+
-
 
+
-
#tabsE a {
+
       float:left;
       float:left;
-
       background:url("tableftE.gif") no-repeat left top;
+
       background:url("tableft10.gif") no-repeat left top;
       margin:0;
       margin:0;
       padding:0 0 0 2px;
       padding:0 0 0 2px;
       text-decoration:none;
       text-decoration:none;
       }
       }
-
     #tabsE a span {
+
     #tabs10 a span {
       float:left;
       float:left;
       display:block;
       display:block;
-
       background:url("tabrightE.gif") no-repeat right top;
+
       background:url("tabright10.gif") no-repeat right top;
       padding:5px 15px 4px 6px;
       padding:5px 15px 4px 6px;
       color:#FFF;
       color:#FFF;
       }
       }
     /* Commented Backslash Hack hides rule from IE5-Mac \*/
     /* Commented Backslash Hack hides rule from IE5-Mac \*/
-
     #tabsE a span {float:none;}
+
     #tabs10 a span {float:none;}
     /* End IE5-Mac hack */
     /* End IE5-Mac hack */
-
     #tabsE a:hover span {
+
     #tabs9 a:hover span {
-
       color:#FFF;
+
       color:#FFFFFF;
       }
       }
-
     #tabsE a:hover {
+
     #tabs10 a:hover {
       background-position:0% -42px;
       background-position:0% -42px;
       }
       }
-
     #tabsE a:hover span {
+
     #tabs10 a:hover span {
       background-position:100% -42px;
       background-position:100% -42px;
       }
       }
-
        #tabsE #current a {
+
      #tabs10 #current a {
-
                background-position:0% -42px;
+
              background-position:0% -42px;
-
        }
+
      }
-
        #tabsE #current a span {
+
      #tabs10 #current a span {
-
                background-position:100% -42px;
+
              background-position:100% -42px;
-
        }
+
      }
-
sfHover = function() {
+
-
var sfEls = document.getElementById("tabsE").getElementsByTagName("LI");
+
-
for (var i=0; i<sfEls.length; i++) {
+
-
sfEls[i].onmouseover=function() {
+
-
this.className+=" sfhover";
+
-
}
+
-
sfEls[i].onmouseout=function() {
+
-
this.className=this.className.replace(new RegExp(" sfhover\\b"), "");
+
-
}
+
-
}
+
-
}
+
-
if (window.attachEvent) window.attachEvent("onload", sfHover);
+
-
 
+
-->
-->
</style>
</style>
-
                </head>
+
        </head>
-
                <body>
+
        <body>
-
                        <div id="tabsE">
+
                <div id="tabs10">
-
                                <ul>
+
                <ul>
-
                                        <!-- CSS Tabs -->
+
                                <!-- CSS Tabs -->
<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a>
+
<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a></li>
-
<ul style="z-index:1"> <br>
+
<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a></li>
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Team">Team</a></li> <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Member">Member</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/School">School</a></li>
+
-
</ul>
+
-
</li>
+
-
<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a>
+
-
<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Project">Overall Project</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_1">Circuit 1</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_2">Circuit 2</a></li>
+
-
</ul>
+
-
</li>
+
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Modeling"><span>Modeling</span></a></li>
+
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Result"><span>Result</span></a>
+
-
<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid1">Plasmid 1</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid2">Plasmid 2</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/lux_promoter">lux Promoter</a></li><br>
+
-
</ul>
+
-
</li>
+
-
<li id="current"><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
+
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
 +
<li id="current"><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
 +
 +
                        </ul>
 +
                </div>
 +
<!--- 1st tab End --->
 +
<!--- 2nd tab --->
 +
<!--- 2nd tab --->
 +
<div id="search-controls">
 +
<form action="/Special:Search" id="searchform">
 +
<input id="searchInput" name="search" type="text" title="Search 2010.igem.org [f]" accesskey="f" value="" />
 +
<input type='submit' name="go" class="searchButton" id="searchGoButton" value="Go" title="Go to a page with this exact name if exists" />&nbsp;
 +
      <input type='submit' name="fulltext" class="searchButton" id="mw-searchButton" value="Search" title="Search the pages for this text" />
 +
</form>
 +
</div> <!-- close search controls -->
 +
<p>
 +
<style type="text/css">
 +
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
 +
<html xmlns="http://www.w3.org/1999/xhtml">
 +
        <head>
 +
                <meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
 +
                <title>Free CSS Navigation Menu Designs 2 at exploding-boy.com</title>
 +
                <style type="text/css">
 +
<!--
 +
    body {
 +
        margin:0;
 +
        padding:0;
 +
        font: bold 11px/1.5em Verdana;
 +
}
-
                                </ul>
+
h2 {
-
                         </div>
+
        font: bold 14px Verdana, Arial, Helvetica, sans-serif;
-
                 </body>
+
        color: #000;
-
</html>
+
        margin: 0px;
 +
        padding: 0px 0px 0px 15px;
 +
}
 +
 
 +
img {
 +
border: none;
 +
}
 +
 
 +
/*- Menu Tabs 9--------------------------- */
 +
 
 +
    #tabs9 {
 +
      float:left;
 +
      width:100%;
 +
      font-size:100%;
 +
      line-height:normal;
 +
      background:#125FDB;
 +
      margin-top:0px;
 +
      }
 +
    #tabs9 ul {
 +
          margin:0;
 +
          padding:2px 10px 0 105px;
 +
          list-style:none;
 +
      }
 +
    #tabs9 li {
 +
      float:left;
 +
      display:inline;
 +
      margin:0;
 +
      padding:0;
 +
      margin-left:40px;
 +
      }
 +
    #tabs9 a {
 +
      float:left;
 +
      background:url("tableft10.gif") no-repeat left top;
 +
      margin:0;
 +
      padding:0 0 0 2px;
 +
      text-decoration:none;
 +
      }
 +
    #tabs9 a span {
 +
      float:left;
 +
      display:block;
 +
      background:url("tabright10.gif") no-repeat right top;
 +
      padding:5px 15px 4px 6px;
 +
      color:#FFF;
 +
      }
 +
    /* Commented Backslash Hack hides rule from IE5-Mac \*/
 +
    #tabs9 a span {float:none;}
 +
    /* End IE5-Mac hack */
 +
    #tabs9 a:hover span {
 +
      color:#FFFFFF;
 +
      }
 +
    #tabs9 a:hover {
 +
      background-position:0% -42px;
 +
      }
 +
    #tabs9 a:hover span {
 +
      background-position:100% -42px;
 +
      }
 +
 
 +
      #tabs9 #current a {
 +
              background-position:0% -42px;
 +
      }
 +
      #tabs9 #current a span {
 +
              background-position:100% -42px;
 +
      }
 +
-->
 +
</style>
 +
        </head>
 +
 
 +
        <body>
 +
                <div id="tabs9">
 +
                <ul>
 +
                                <!-- CSS Tabs -->
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook 1</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_2"><span>Notebook 2</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_3"><span>Notebook 3</span></a></li>
 +
                         </ul>
 +
                 </div>
 +
        </body>
 +
<!--- 2nd tab End--->
<!--- Background Contents End --->
<!--- Background Contents End --->
<!--- Main Contents Start --->
<!--- Main Contents Start --->
-
<br><br>
+
<br><br><br>
<html><h3>2010-08-31</h3></html>
<html><h3>2010-08-31</h3></html>
Making plate with Broth
Making plate with Broth
Line 179: Line 263:
<html><h3>2010-09-06</h3></html>
<html><h3>2010-09-06</h3></html>
Co-transformation
Co-transformation
-
  Plux – gfp + Pc each 1㎕
+
  Plux – gfp + Pc each 1uL
-
  SOC 200㎕
+
  SOC 200uL
<html><h3>2010-09-07</h3></html>
<html><h3>2010-09-07</h3></html>
Line 187: Line 271:
<html><h3>2010-09-08</h3></html>
<html><h3>2010-09-08</h3></html>
Transformation
Transformation
-
  XL10GkanR 30㎕ DNA 1㎕ SOC 100㎕
+
  XL10GkanR 30uL DNA 1uL SOC 100uL
  K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
  K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
<html><h3>2010-09-14</h3></html>
<html><h3>2010-09-14</h3></html>
  1) K091204(2-8J) Pc – luxR function check
  1) K091204(2-8J) Pc – luxR function check
-
   [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1㎕ + XL10GkanR 50㎕
+
   [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1uL + XL10GkanR 50uL
   AHL : 30C8HSL(1/10000 of 1μM)
   AHL : 30C8HSL(1/10000 of 1μM)
  2) T9002  
  2) T9002  
Line 201: Line 285:
               (1)      (2)
               (1)      (2)
   --------------------------------
   --------------------------------
-
     template  1㎕
+
     template  1uL
-
     primer    5㎕
+
     primer    5uL
-
     F          5㎕
+
     F          5uL
-
     R          5㎕
+
     R          5uL
-
     10x buffer 5㎕
+
     10x buffer 5uL
-
     dNTP      5㎕
+
     dNTP      5uL
-
     NFW        28㎕
+
     NFW        28uL
-
     ventP      1㎕
+
     ventP      1uL
   -------------------------------
   -------------------------------
-
               50㎕     50㎕
+
               50uL     50uL
   Transformation of BBa_J04450(3A) -> incubation
   Transformation of BBa_J04450(3A) -> incubation
Line 231: Line 315:
2. Same to mini-prep
2. Same to mini-prep
-
   * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
+
   * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
-
   * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
+
   * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
-
3. NFW elution(10㎕)
+
3. NFW elution(10uL)
Add NFW and wait 1min. Operate centrifuge in 1min
Add NFW and wait 1min. Operate centrifuge in 1min
-
4. Gel electrophoresis(each 1㎕)
+
4. Gel electrophoresis(each 1uL)
  SOEing PCR
  SOEing PCR
Line 245: Line 329:
   ⑤Prom-luxR-(OR2)
   ⑤Prom-luxR-(OR2)
   ⑥Prom-luxR-(OR1)
   ⑥Prom-luxR-(OR1)
-
  1. ①1㎕, ③3㎕
+
  1. ①1uL, ③3uL
-
  2. ②1㎕, ④4㎕
+
  2. ②1uL, ④4uL
-
  3. ①1㎕, ⑤1㎕
+
  3. ①1uL, ⑤1uL
-
  4. ②1㎕, ⑥1㎕
+
  4. ②1uL, ⑥1uL
  1. ①-③
  1. ①-③
  2. ②-④
  2. ②-④
   --------------------------
   --------------------------
-
   template    1㎕-4㎕
+
   template    1uL-4uL
   primer Fwd  -
   primer Fwd  -
   primer Rev  -
   primer Rev  -
-
   dNTP        5㎕
+
   dNTP        5uL
-
   10xbuffer  5㎕
+
   10xbuffer  5uL
-
   NFW        34㎕
+
   NFW        34uL
-
   VentP      1㎕
+
   VentP      1uL
   -------------------------
   -------------------------
-
               50㎕<br>
+
               50uL<br>
  3. ①-⑤
  3. ①-⑤
  4. ②-⑥
  4. ②-⑥
   -------------------------
   -------------------------
-
   template      1㎕-1㎕
+
   template      1uL-1uL
   primer Fwd    -
   primer Fwd    -
   primer Rev    -
   primer Rev    -
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer    5㎕
+
   10xbuffer    5uL
-
   NFW          37㎕
+
   NFW          37uL
-
   VentP        1㎕
+
   VentP        1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  5. control
  5. control
   -------------------------
   -------------------------
-
   template      1㎕
+
   template      1uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer    5㎕
+
   10xbuffer    5uL
-
   NFW          28㎕
+
   NFW          28uL
-
   VentP        1㎕
+
   VentP        1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  PCR
  PCR
   94°C – 5min
   94°C – 5min
Line 293: Line 377:
   ----------------
   ----------------
   4°C  stop
   4°C  stop
-
 
<html><h3>2010-09-20</h3></html>
<html><h3>2010-09-20</h3></html>
Line 299: Line 382:
  BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
  BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
  2-1 P    pSB1AK3
  2-1 P    pSB1AK3
-
  11:30 ~  DNA : 1㎕   XL10GkanR : 30㎕   SOC : 100㎕<br>
+
  11:30 ~  DNA : 1uL   XL10GkanR : 30uL   SOC : 100uL<br>
  Cm              Amp
  Cm              Amp
  1-3C            1-1K
  1-3C            1-1K
Line 308: Line 391:
ZYMO purification
ZYMO purification
  From PCR product from SOEing PCR (09-19)
  From PCR product from SOEing PCR (09-19)
-
  1. Add PCR products(50㎕) and DNA binding buffer(200㎕) into 1.5mL tube(Commonly adding DNA binding buffer for double
+
  1. Add PCR products(50uL) and DNA binding buffer(200uL) into 1.5mL tube(Commonly adding DNA binding buffer for double
-
  amount of PCR products, but it must be 200㎕ at least). After that, mix this tube for a second with vortex and move
+
  amount of PCR products, but it must be 200uL at least). After that, mix this tube for a second with vortex and move
  to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
  to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
  2. centrifuge 1min → throwing away left solution
  2. centrifuge 1min → throwing away left solution
  3. Same to mini-prep
  3. Same to mini-prep
-
   * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
+
   * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
-
   * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
+
   * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
-
  4. NFW elution(10㎕)
+
  4. NFW elution(10uL)
↓<br>
↓<br>
PCR
PCR
Line 329: Line 412:
  After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
  After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
   -------------------------
   -------------------------
-
   template      10㎕
+
   template      10uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer      5㎕
+
   10xbuffer      5uL
-
   NFW            19㎕
+
   NFW            19uL
-
   VentP          1㎕
+
   VentP          1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  Using primer
  Using primer
   1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
   1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
Line 365: Line 448:
Liquid Incubation(37°C) 7:15~
Liquid Incubation(37°C) 7:15~
  -------------------------
  -------------------------
-
  template      3㎕
+
  template      3uL
-
  primer Fwd    5㎕
+
  primer Fwd    5uL
-
  primer Rev    5㎕
+
  primer Rev    5uL
-
  dNTP          5㎕
+
  dNTP          5uL
-
  10xbuffer      5㎕
+
  10xbuffer      5uL
-
  NFW            26㎕
+
  NFW            26uL
-
  VentP          1㎕
+
  VentP          1uL
  -------------------------
  -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  Using primer
  Using primer
   Fwd : pSB1C3FA-F
   Fwd : pSB1C3FA-F
Line 391: Line 474:
↓<br>
↓<br>
Gel Electrophoresis
Gel Electrophoresis
-
 
-
 
-
<html><h3>2010-09-21</h3></html>
 
-
PCR
 
-
Because it didn’t come out vector PCR(gel electrophoresis),
 
-
we operated re-experiment to change template and template amounts.<br>
 
-
Using template
 
-
  1. Biobrick 2010 1-3A(pSB1C3)
 
-
  2. Biobrick 2010 pSB1C3
 
-
  3. Biobrick 2010 2-9A(pSB1A3)
 
-
  4. control<br>
 
-
Using primer
 
-
  Fwd : pSB1C3 FASTR-F
 
-
  Rev : pSB1C3 FASTR-R<br>
 
-
  -------------------------
 
-
  template      5㎕
 
-
  primer Fwd    5㎕
 
-
  primer Rev    5㎕
 
-
  dNTP          5㎕
 
-
  10xbuffer      5㎕
 
-
  NFW            24㎕
 
-
  VentP          1㎕
 
-
  -------------------------
 
-
                50㎕<br>
 
-
PCR(TAKARA)
 
-
  94°C – 5min
 
-
  94°C – 15sec    --
 
-
  51°C – 30sec      │- 25 cycles
 
-
  72°C – 1min    --
 
-
  72°C – 10min
 
-
 
-
Vector PCR
 
-
Using template
 
-
  1. Biobrick 2010 1-3A(pSB1C3)
 
-
  2. Biobrick 2010 pSB1C3
 
-
  3. Biobrick 2010 2-9A(pSB1A3)
 
-
  4. control<br>
 
-
  -------------------------
 
-
  template      3㎕
 
-
  primer Fwd    5㎕
 
-
  primer Rev    5㎕
 
-
  dNTP          5㎕
 
-
  10xbuffer    5㎕
 
-
  NFW          26㎕
 
-
  VentP        1㎕
 
-
  -------------------------
 
-
                50㎕<br>
 
-
Using primer
 
-
  Fwd : pSB1C3 FASTR-F
 
-
  Rev : pSB1C3 FASTR-R<br>
 
-
PCR Condition
 
-
  94°C – 5min
 
-
  94°C – 30sec      --
 
-
  51°C – 30sec        │-25 cycles
 
-
  72°C – 2min      --
 
-
  72°C – 10min
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<html><h3>2010-09-22</h3></html>
 
-
  Biobrick 2010 2-9A(pSB1A3) -> gel extraction
 
-
 
-
gel electrophoresis - Biobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3),
 
-
Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3)
 
-
  As a result, it didn’t come out band of 1-3A and pSB1C3. We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.
 
-
 
-
  FASTR Cloning (Plasmid1)
 
-
  Vector (about 2kbp)
 
-
- pSB1A3 (conservative solution 100ng/㎕)
 
-
  Insert (about 1kbp)
 
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
 
-
master mix
 
-
---------------------------------------------------------------------
 
-
Vector(50ng)        0.5 2
 
-
Insert(75ng) 3 -
 
-
Tango buffer 0.5 2
 
-
T4 Ligase buffer 0.5 2
 
-
ATP 1 4
 
-
LguI 0.5 2
 
-
Ligase 0.5 2
 
-
DpnI 0.5 2
 
-
NFW 3 12
 
-
-----------------------------------------------------------------------
 
-
Total 10㎕ 28㎕
 
-
Room temperature 2h
 
-
 
-
  Transformation
 
-
  To use cell strain : BL21(DE3) 50㎕
 
-
  To use plasmid
 
-
      L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
 
-
      L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
 
-
      L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
 
-
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
 
-
 
-
  After transformation
 
-
    We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
 
-
 
-
  ※A Reason of using DE3 cell strain
 
-
DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed. Plasmid1 has T7/cI hybrid promoter. So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.
 
-
 
-
<html><h3>2010-09-23</h3></html>
 
-
  From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50㎕ Elution Solution.
 
-
  ↓
 
-
gel electrophoresis(1㎕)
 
-
 
-
 
-
L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
 
-
    L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
 
-
    L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
 
-
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
 
-
 
-
Remaining of above sample is transformed and incubated.
 
-
cell strain : XL10G(50㎕/tube)
 
-
plasmid : L1~L4(total 4)
 
-
 
 
-
FASTR Cloning (Plasmid1)
 
-
  Vector (about 2kbp)
 
-
- pSB1A3 (conservative solution 100ng/㎕)
 
-
  Insert (about 1kbp)
 
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
 
-
4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
 
-
 
-
master mix
 
-
---------------------------------------------------------------------
 
-
Vector(50ng)        0.5 2
 
-
Insert(75ng) 3 -
 
-
Tango buffer 0.5 2
 
-
T4 Ligase buffer 0.5 2
 
-
ATP 1 4
 
-
LguI 0.5 2
 
-
Ligase 0.5 2
 
-
DpnI 0.5 2
 
-
NFW 3 12
 
-
-----------------------------------------------------------------------
 
-
Total 10㎕ 28㎕
 
-
Room temperature 2h
 
-
 
-
After ligation plasmid is transformed and incubated in LB broth.
 
-
cell strain : BL21(DE3), XL10G(each 50㎕/tube)
 
-
plasmid : L1~L4
 
-
 
-
 
-
 
-
 
-
<html><h3>2010-09-24</h3></html>
 
-
 
-
At 23th September
 
-
Remaining of above sample is transformed and incubated.
 
-
cell strain : XL10G(50㎕/tube)
 
-
plasmid : L1~L4(total 4)
 
-
 
-
We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.
 
-
 
-
Insert Check Colony PCR(Plasmid 1)
 
-
master mix
 
-
--------------------------------------------------------------------------
 
-
Template(picked colonies)
 
-
Primer VF 2㎕ 40㎕
 
-
Primer VR 2㎕ 40㎕
 
-
10x Buffer 2㎕ 40㎕
 
-
dNTP 2㎕ 40㎕
 
-
NFW 10㎕ 200㎕
 
-
Taq DNA Polymerase 0.5㎕ 5㎕
 
-
--------------------------------------------------------------------------
 
-
Total 20㎕ 400㎕
 
-
 
-
↓
 
-
PCR
 
-
94°C – 5min
 
-
94°C – 30sec
 
-
51°C – 30sec        20 cycles
 
-
72°C – 1min
 
-
72°C – 10min
 
-
↓
 
-
gel electrophoresis
 
-
 
-
 
-
2010-09-25
 
-
mini-prep and gel electrophoresis of liquid culture medium at 24th September
 
-
 
-
gel electrophoresis picture
 
-
↓
 
-
Transformation to BL21(DE3)
 
-
↓
 
-
Incubation in LB broth, IPTG(0, 10, 100 μM)
 
-
↓37°C
 
-
About total sample, it is shown GFP ON/OFF switching on eyes.
 
-
↓
 
-
For the purpose of measuring fluorescence level, we did liquid incubation from each plate.
 
-
IPTG(0, 10, 100 μM) x 6 samples = 18 test tubes
 
-
↓37°C
 
-
∙ measuring fluorescence level(liquid incubation)
 
-
∙ pelletization of cell
 
-
 
-
 
-
Digestion and ligation of BBa-J01011(Ptet-cI) and pSB3C5
 
-
(Digestion)
 
-
Insert(J01011)         Vector(pSB3C5)
 
-
DNA 10㎕       10㎕
 
-
NFW 32.5㎕       32.5㎕
 
-
NEB Buffer 2 5㎕     5㎕
 
-
BSA 0.5㎕       0.5㎕
 
-
EcoRI 1㎕     1㎕
 
-
PstI 1㎕     1㎕
 
-
------------------------------------------------------------------------
 
-
total                      50㎕                50㎕
 
-
↓37°C 15min incubator
 
-
↓65°C 20min (sterilization dryer)
 
-
 
-
※After digestion, we did gel electrophoresis
 
-
 
-
(Ligation)
 
-
  13㎕ NFW
 
-
    2㎕ Insert(digested)
 
-
    2㎕ Vector(digested)                  total 20㎕ in 0.2mL PCR tube
 
-
    2㎕ 10x T4 DNA Ligase Buffer
 
-
    1㎕ T4 DNA Ligase
 
-
 
-
↓10min at room temperature
 
-
↓20min at 65°C (sterilization dryer)
 
-
 
-
  (Transformation)
 
-
  Transformation XL10G 50㎕ to 10㎕ of ligation product
 
-
↓37°C
 
-
  Insert check of living colonies(colony PCR)    /    liquid incubation
 
-
          ↓total 7 colonies                              ↓37°C
 
-
  Insert Check Colony PCR(Plasmid 1)                     ↓We did mini-prep only No.4 sample checked insert.
 
-
  --------------------------------------------
 
-
Template(picked colonies)                                  ↓
 
-
Primer VF 2㎕                                     Gel electrophoresis
 
-
Primer VR 2㎕
 
-
10x Buffer 2㎕
 
-
dNTP 2㎕
 
-
NFW 11.5㎕
 
-
Taq DNA Polymerase 0.5㎕
 
-
--------------------------------------------------------
 
-
Total 20㎕
 
-
↓
 
-
PCR
 
-
  94°C 5min
 
-
  94°C 30sec
 
-
  51°C 30sec    30 cycle
 
-
  72°C 1min
 
-
  72°C 10min
 
-
  ↓
 
-
Gel electrophoresis
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
==Notebook==
==Notebook==

Latest revision as of 19:10, 27 October 2010




 

 




2010-08-31

Making plate with Broth 

Amp x 13 
Kan x 1 
Amp + IPTG x 1

Transformation 

R0010 B0030 B0032 B0033 K145270 I746903 R0051 C0051 K145001 C0012 K145001 C0012 P0312 I719015

2010-09-02

Mini-prep(DNA transformated in August 31th) 

I13522 C0040 C0052 C0053 C0080 C0072 I0500 Q04510 Q04400 Q04121 R0040 B0014
B1101 I715038 K081012 K082034 K113007 I763011

2010-09-03

Preparing for experiment 

We inserted plasmid with cI promoter + GFP to E.coli. And

2010-09-04

Preparing for experiment 

I746903

2010-09-05

K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801

2010-09-06

Co-transformation 

Plux – gfp + Pc each 1uL
SOC 200uL

2010-09-07

E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121

2010-09-08

Transformation 

XL10GkanR 30uL DNA 1uL SOC 100uL
K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034

2010-09-14

1) K091204(2-8J) Pc – luxR function check
  [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1uL + XL10GkanR 50uL
  AHL : 30C8HSL(1/10000 of 1μM)
2) T9002

2010-09-16

 PCR
  T7/cI(OR1/2) – GFP 
              (1)      (2)
  --------------------------------
   template   1uL
   primer     5uL
   F          5uL
   R          5uL
   10x buffer 5uL
   dNTP       5uL
   NFW        28uL
   ventP      1uL
  -------------------------------
             50uL     50uL
 Transformation of BBa_J04450(3A) -> incubation

2010-09-19

From 2010 Biobrick
 plasmid with Cm antibiotic   -------------
  1-3C 1-3A pSB1C3
 plasmid with Amp antibiotic  ------------- 
  1-1K pSB1A3
 ↓
To transformate total five types, we seeded to plates.
 ↓
PCR products
 ↓
Gel extraction
 ↓  ← ADB buffer
ZYMO purification

1. To prepare 2ml tube(for ZYMO purification) + column, add the solution and operate centrifuge in 1 minute. Throw away left solution.

2. Same to mini-prep 

 * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1

3. NFW elution(10uL) Add NFW and wait 1min. Operate centrifuge in 1min 4. Gel electrophoresis(each 1uL) 

SOEing PCR
 ①T7/cI(OR2)-F-R
 ②T7/cI(OR1)-F-R
 ③Ptet-luxR-(OR2)
 ④Ptet-luxR-(OR1)
 ⑤Prom-luxR-(OR2)
 ⑥Prom-luxR-(OR1)
1. ①1uL, ③3uL
2. ②1uL, ④4uL
3. ①1uL, ⑤1uL
4. ②1uL, ⑥1uL

1. ①-③
2. ②-④
 --------------------------
 template    1uL-4uL
 primer Fwd  -
 primer Rev  -
 dNTP        5uL
 10xbuffer   5uL
 NFW         34uL
 VentP       1uL
 -------------------------
             50uL

3. ①-⑤
4. ②-⑥
 -------------------------
 template      1uL-1uL
 primer Fwd    -
 primer Rev    -
 dNTP          5uL
 10xbuffer     5uL
 NFW           37uL
 VentP         1uL
 -------------------------
               50uL

5. control
 -------------------------
 template      1uL
 primer Fwd    5uL
 primer Rev    5uL
 dNTP          5uL
 10xbuffer     5uL
 NFW           28uL
 VentP         1uL
 -------------------------
               50uL

PCR
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │- 10cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

2010-09-20

Transformation 

BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
2-1 P    pSB1AK3
11:30 ~   DNA : 1uL    XL10GkanR : 30uL    SOC : 100uL

Cm              Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

liquid incubation(37°C) 7:15~
Mini-prep

ZYMO purification 

From PCR product from SOEing PCR (09-19)
1. Add PCR products(50uL) and DNA binding buffer(200uL) into 1.5mL tube(Commonly adding DNA binding buffer for double
amount of PCR products, but it must be 200uL at least). After that, mix this tube for a second with vortex and move
to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
2. centrifuge 1min → throwing away left solution
3. Same to mini-prep
 * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
 * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
4. NFW elution(10uL)

↓
PCR 

PCR Condition
 94°C – 5min
 94°C – 15sec     --
 52°C – 30sec       │-10 cycles
 72°C – 1min      --
 72°C – 10min
 ----------------
 4°C   stop

After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
 -------------------------
 template      10uL
 primer Fwd     5uL
 primer Rev     5uL
 dNTP           5uL
 10xbuffer      5uL
 NFW            19uL
 VentP          1uL
 -------------------------
                50uL

Using primer
 1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
 3. ①-⑤ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 4. ②-⑥ Fwd : PluxR∙FA-R     Rev : TcIg∙FA-R
 5. control

Using template
 1. Ptet-LuxR-PT7/cI(OR2)-GFP
 2. Ptet-LuxR-PT7/cI(OR1)-GFP
 3. Prom-LuxR-Pt7/cI(OR2)-GFP
 4. Prom-LuxR-Pt7.cI(OR1)-GFP
 5. control

PCR condition(usingTAKARA PCR thermal cycler)
 94°C – 5min
 94°C – 15sec     --
 51°C – 30sec       │-25 cycles
 72°C – 1min      --
 72°C – 10min

↓
Gel Electrophoresis 

Cm             Amp
1-3C            1-1K
1-3A            pSB1A3
pSB1C3

↓
Liquid Incubation(37°C) 7:15~ 

-------------------------
template       3uL
primer Fwd     5uL
primer Rev     5uL
dNTP           5uL
10xbuffer      5uL
NFW            26uL
VentP          1uL
-------------------------
               50uL

Using primer
 Fwd : pSB1C3FA-F
 Rev : pSB1C3FA-R

Using template
 1. 1-3C(J04450,pSB3C5)
 2. pSB1C3
 3. 1-3A(J04450,pSB1C3)
 4. 1-1K(J04450,J63010)
 5. control(pCI-GFP)   primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev

PCR condition
 94°C – 5min
 94°C – 15sec    --
 51°C – 30sec      │-25 cycles
 72°C – 1min     --
 72°C – 10min

↓
Gel Electrophoresis

Notebook

8/31 We've started experiment. First, we researched the property of T7 promoter.

9/5 We are researching promoter and inverters.

10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D
Retrieved from "http://2010.igem.org/Team:Chiba/Notebook_1"