Team:Washington/GENERAL PCR PROTOCOL

From 2013.igem.org

(Difference between revisions)
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<html>
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{{:Team:Washington/WA_Template}}
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<style type="text/css">
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.firstHeading {                                                   /* Gets rid of the default iGEM picture*/
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    height:2px;
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    visibility:hidden;
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}
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#catlinks {
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    display: none;
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}
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#content {
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    border-left-width:0px;
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    border-right-width:0px;
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    padding: 0px 5px 0px 5px;
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    width:965px;                                                    /* Content width*/
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}
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#menubar {
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    background-color:  #FFFFFF;                              /* left menu bar color*/
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}
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#menubar ul li a {
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    color: #545353; }                                            /* menu text color*/
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    .right-menu li a {
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    color: red; 
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    background-color: #FFFFFF;                              /* right menu bar color*/
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}
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#p-logo { height 1px; overflow:hidden; display: none;
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}
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#top-section {     
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        background-position: center center;
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        background-repeat: no-repeat;
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        background-color: #FFFFFF;                        /* top header background color*/
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        border-width:0px;
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        height:10px;                        /* top header height (determines how low content will start to appear)*/
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}
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#siteSub {
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display:none;
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}
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#search-controls {                                                /* gets rid of the search bar (it was ugly) */
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display:none;
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margin-top:0px;
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}
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#contentSub {
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display:none;
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}
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/*Now to set heading and body fonts*/
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body {
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background:#4B0082;
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background-image: url("https://static.igem.org/mediawiki/2012/1/1b/Gradientback.jpg");
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background-position: center;
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background-repeat: repeat-y;
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}               
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h1
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{
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color:#262626;
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text-align:left;
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font-family:"Trebuchet";
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font-size:25px;
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line-height:100%;
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border-bottom: none;
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padding:0px 0px 0px 0px; 
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margin: 0px 0px 0px 0px;
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}
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h2
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{
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color:#262626;
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text-align:left;
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font-family:"Trebuchet";
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font-size:20px;
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line-height: 100%;
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border-bottom: none; 
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padding:0px 0px 0px 0px; 
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margin: 0px 0px 0px 0x;
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}
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h3
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{
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color:#262626;
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text-align:left;
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font-family:"Trebuchet";
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font-size:17px;
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line-height: 100%;
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border-bottom: none; 
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padding:0px 0px 0px 0px; 
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margin: 0px 0px 0px 0x;
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}
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p
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{
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color:#262626;
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text-align: justify;
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font-family:"Arial";
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font-size:14px;
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line-height:100%
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border-bottom: none;
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padding:0px 0px 0px 0px; 
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margin: 0px 0px 0px 0x;
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border:0px
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}
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body {
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color:#808080;
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line-height: 1;
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font-family:"Arial";
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font-size:11px; 
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}
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.whitebox {
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  -moz-box-shadow:  0px 0px 0px #ccc;
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  -webkit-box-shadow:  0px 0px 0px #ccc;
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  box-shadow:  0px 0px 15px #ccc;
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  background-color: #FFFFFF;
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  -moz-border-radius: 1px;
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  border-radius: 15px;
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  margin-top: 20px;
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  padding-top: 20px;
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  padding-left: 20px;
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  padding-right: 20px;
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  padding-bottom: 20px;
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  margin-left: 15px;
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  margin-right: 25px;
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}
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.thumbnail
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{
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margin-bottom:0px;
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padding-bottom:0px;
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position:relative;
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}
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#igemuwmenu, #igemuwmenu ul {
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margin: 0;
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padding: 0;
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list-style: none;
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}
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#igemuwmenu {
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width: 820px;
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margin: 10px auto;
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border: 1px solid #222;
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background-color: #111;
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background-image: -moz-linear-gradient(#444, #111);
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background-image: -webkit-gradient(linear, left top, left bottom, from(#444), to(#111));
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background-image: -webkit-linear-gradient(#444, #111);
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background-image: -o-linear-gradient(#444, #111);
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background-image: -ms-linear-gradient(#444, #111);
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background-image: linear-gradient(#444, #111);
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-moz-border-radius: 6px;
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-webkit-border-radius: 6px;
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border-radius: 6px;
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-moz-box-shadow: 0 1px 1px #777, 0 1px 0 #666 inset;
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-webkit-box-shadow: 0 1px 1px #777, 0 1px 0 #666 inset;
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box-shadow: 0 1px 1px #777, 0 1px 0 #666 inset;
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                display: inline-block;
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                white-space: nowrap;
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}
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#igemuwmenu:before,
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#igemuwmenu:after {
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content: "";
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display: table;
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}
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#igemuwmenu:after {
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clear: both;
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}
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#igemuwmenu {
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zoom:1;
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}
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#igemuwmenu li {
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border-right: 1px solid #222;
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-moz-box-shadow: 1px 0 0 #444;
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-webkit-box-shadow: 1px 0 0 #444;
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box-shadow: 1px 0 0 #444;
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position: relative;
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                display: inline-block;
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                white-space: nowrap;
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}
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#igemuwmenu a {
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padding: 12px 20px;
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color: #999;
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text-transform: uppercase;
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font: bold 11.4px Arial, Helvetica;
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text-decoration: none;
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text-shadow: 0 1px 0 #000;
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                display: inline-block;
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                white-block: nowrap;
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}
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    #igemuwmenu li:hover > a {
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color: #fafafa;
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}
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*html #igemuwmenu li a:hover { /* IE6 only */
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color: #fafafa;
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}
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#igemuwmenu ul {
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margin: 20px 0 0 0;
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_margin: 0; /*IE6 only*/
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opacity: 0;
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visibility: hidden;
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position: absolute;
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top: 38px;
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left: 0;
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z-index: 1;   
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background: #444;
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background: -moz-linear-gradient(#444, #111);
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background-image: -webkit-gradient(linear, left top, left bottom, from(#444), to(#111));
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background: -webkit-linear-gradient(#444, #111);   
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background: -o-linear-gradient(#444, #111);
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background: -ms-linear-gradient(#444, #111);
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background: linear-gradient(#444, #111);
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-moz-box-shadow: 0 -1px rgba(255,255,255,.3);
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-webkit-box-shadow: 0 -1px 0 rgba(255,255,255,.3);
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box-shadow: 0 -1px 0 rgba(255,255,255,.3);
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-moz-border-radius: 3px;
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-webkit-border-radius: 3px;
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border-radius: 3px;
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-webkit-transition: all .2s ease-in-out;
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-moz-transition: all .2s ease-in-out;
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-ms-transition: all .2s ease-in-out;
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-o-transition: all .2s ease-in-out;
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transition: all .2s ease-in-out; 
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}
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#igemuwmenu li:hover > ul {
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opacity: 1;
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visibility: visible;
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margin: 0;
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}
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#igemuwmenu ul ul {
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top: 0;
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left: 150px;
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margin: 0 0 0 20px;
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_margin: 0; /*IE6 only*/
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-moz-box-shadow: -1px 0 0 rgba(255,255,255,.3);
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-webkit-box-shadow: -1px 0 0 rgba(255,255,255,.3);
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box-shadow: -1px 0 0 rgba(255,255,255,.3);
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}
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#igemuwmenu ul li {
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float: none;
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display: block;
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border: 0;
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_line-height: 0; /*IE6 only*/
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-moz-box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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-webkit-box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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}
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#igemuwmenu ul li.active {
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float: none;
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display: block;
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border: 0;
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_line-height: 0; /*IE6 only*/
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-moz-box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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-webkit-box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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box-shadow: 0 1px 0 #111, 0 2px 0 #666;
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}
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#igemuwmenu ul li:last-child { 
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-moz-box-shadow: none;
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-webkit-box-shadow: none;
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box-shadow: none;   
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}
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-
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#igemuwmenu ul a {   
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padding: 10px;
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width: 130px;
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_height: 10px; /*IE6 only*/
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display: block;
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white-space: nowrap;
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float: none;
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text-transform: none;
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}
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-
     
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        #igemuwmenu ul a.selected {   
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padding: 10px;
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width: 130px;
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_height: 10px; /*IE6 only*/
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display: block;
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white-space: nowrap;
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float: none;
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text-transform: none;
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}
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#igemuwmenu ul a:hover {
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background-color: #785D84;
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background-image: -moz-linear-gradient(#B6A0BA,  #785D84);
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background-image: -webkit-gradient(linear, left top, left bottom, from(#B6A0BA), to(#785D84));
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background-image: -webkit-linear-gradient(#B6A0BA, #785D84);
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background-image: -o-linear-gradient(#B6A0BA, #785D84);
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background-image: -ms-linear-gradient(#B6A0BA, #785D84);
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background-image: linear-gradient(#B6A0BA, #785D84);
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}
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#igemuwmenu ul li:first-child > a {
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-moz-border-radius: 3px 3px 0 0;
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-webkit-border-radius: 3px 3px 0 0;
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border-radius: 3px 3px 0 0;
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}
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#igemuwmenu ul li:first-child > a:after {
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content: '';
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position: absolute;
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left: 40px;
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top: -6px;
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border-left: 6px solid transparent;
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border-right: 6px solid transparent;
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border-bottom: 6px solid #444;
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}
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-
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#igemuwmenu ul ul li:first-child a:after {
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left: -6px;
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top: 50%;
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margin-top: -6px;
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border-left: 0;
+
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border-bottom: 6px solid transparent;
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-
border-top: 6px solid transparent;
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-
border-right: 6px solid #3b3b3b;
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-
}
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-
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-
#igemuwmenu ul li:first-child a:hover:after {
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border-bottom-color: #B6A0BA;
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-
}
+
-
+
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#igemuwmenu ul ul li:first-child a:hover:after {
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border-right-color: #0299d3;
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border-bottom-color: transparent;
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}
+
-
+
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#igemuwmenu ul li:last-child > a {
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-moz-border-radius: 0 0 3px 3px;
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-webkit-border-radius: 0 0 3px 3px;
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border-radius: 0 0 3px 3px;
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-
}
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/* Mobile */
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#igemuwmenu-trigger {
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display: none;
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}
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@media screen and (max-width: 600px) {
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/* nav-wrap */
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#igemuwmenu-wrap {
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position: relative;
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}
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#igemuwmenu-wrap * {
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-moz-box-sizing: border-box;
+
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-webkit-box-sizing: border-box;
+
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box-sizing: border-box;
+
-
}
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-
 
+
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/* menu icon */
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#igemuwmenu-trigger {
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display: block; /* show menu icon */
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height: 40px;
+
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line-height: 40px;
+
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cursor: pointer;
+
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padding: 0 0 0 35px;
+
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border: 1px solid #222;
+
-
color: #fafafa;
+
-
font-weight: bold;
+
-
background-color: #111;
+
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background: url(data:image/png;base64,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) no-repeat 10px center, -moz-linear-gradient(#444, #111);
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Revision as of 01:26, 25 September 2013


'

Untitled Document

General PCR Protocol

See also: PCR_GoTaq

Resuspending primers:

Protocol: Resuspending PCR primers


Working Stock:

(serves as a backup)

Dilute to 10 times the number on primer vial

10ul fwd primer 90ul DI water

10ul rev primer 90ul DI water


Notes for iGEM 2013 HSB lab:

We are using the GreenTaq polymerase 2X pre-mixed (GoTaq) for PCR.

Use the protocols below for Green Taq polymerase.

Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).

If PCRing insert use 1ng/ul DNA concentration.


Amplification PCR with Green Taq polymerase:

  1. Setup Amplification PCR Reaction

    1. 1uL Template DNA

    2. 22ul deionized (dI) H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 25ul 2x Green taq (GoTaq)

    6. Total 50ul

  2. Amplification PCR Reaction Program

        1. 95 C for 30s

        2. 95 C for 30s

        3. ZZ* C for 30s (important step)

        4. 72 C for 1min per kb target gene plus 10-15% (important step)

        5. Repeat 2-4 34x

        6. 72 C for 5 to 10min

        7. 4 C for forever (don’t forget to turn the machine off the next day)

  • Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).


Analytical PCR with Green Taq polymerase:

  1. Setup PCR Reaction

    1. 1uL Template

    2. 2ul dI H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 5ul 2x Green taq (GoTaq)

    6. Total 10uls

  2. PCR Reaction Program

        1. 95 C - 30s

        2. 95 C - 10s

        3. ZZ* C - 10s (important step)

        4. 72 C - 1min per kb target gene plus 10-15% (important step)

        5. Repeat b-d 25x

        6. 72 C - 5 to 10min

        7. 10 or 4 C - forever


Phusion polymerase:

  1. Setup Amplification PCR Reaction

    1. 1uL Template

    2. 1uL 25mM dNTP's

    3. 10uL Phusion HF Buffer

    4. 1.0ul Forward Primer (Tm XX oC)*

    5. 1.0ul Reverse Primer (Tm YY oC)*

    6. 0.5uL Phusion polymerase

    7. 36.5uL diH2O

    8. Total 50uls

  2. Amplification PCR Reaction Program

    1. 98 C - 30s

    2. 98 C - 10s

    3. ZZ* C - 10s (important step)

    4. 72 C - 30s per kb target gene plus 10-15% (important step)

    5. Repeat b-d 29-34x

    6. 72 C - 5 to 10min

    7. 10 or 4 C - forever

  • Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).

Refer for further details to NEB's online protocol for Phusion:

Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase

Retrieved from "http://2013.igem.org/Team:Washington/GENERAL_PCR_PROTOCOL"