Team:Washington/PRIMER DESIGN

From 2013.igem.org

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<html>
 +
 +
<style type="text/css">
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.firstHeading {                                                    /* Gets rid of the default iGEM picture*/
 +
    height:2px;
 +
    visibility:hidden;
 +
}
 +
#catlinks {
 +
    display: none;
 +
}
 +
#content {
 +
    border-left-width:0px;
 +
    border-right-width:0px;
 +
    padding: 0px 5px 0px 5px;
 +
    width:965px;                                                    /* Content width*/
 +
}
 +
#menubar {
 +
    background-color:  #FFFFFF;                              /* left menu bar color*/
 +
}
 +
#menubar ul li a {
 +
    color: #545353; }                                            /* menu text color*/
 +
    .right-menu li a {
 +
    color: red; 
 +
    background-color: #FFFFFF;                              /* right menu bar color*/
 +
}
 +
#p-logo { height 1px; overflow:hidden; display: none;
 +
}
 +
#top-section {     
 +
        background-position: center center;
 +
        background-repeat: no-repeat;
 +
        background-color: #FFFFFF;                        /* top header background color*/
 +
        border-width:0px;
 +
        height:10px;                        /* top header height (determines how low content will start to appear)*/
 +
}
 +
#siteSub {
 +
display:none;
 +
}
 +
#search-controls {                                                /* gets rid of the search bar (it was ugly) */
 +
display:none;
 +
margin-top:0px;
 +
}
 +
#contentSub {
 +
display:none;
 +
}
 +
 +
/*Now to set heading and body fonts*/
 +
body {
 +
background:#000000;
 +
background-image: url("https://static.igem.org/mediawiki/2012/1/1b/Gradientback.jpg");
 +
background-position: center;
 +
background-repeat: repeat-y;
 +
}               
 +
h1
 +
{
 +
color:#262626;
 +
text-align:left;
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font-family:"Trebuchet";
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font-size:25px;
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line-height:100%;
 +
border-bottom: none;
 +
padding:0px 0px 0px 0px; 
 +
margin: 0px 0px 0px 0px;
 +
}
 +
h2
 +
{
 +
color:#262626;
 +
text-align:left;
 +
font-family:"Trebuchet";
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font-size:20px;
 +
line-height: 100%;
 +
border-bottom: none; 
 +
padding:0px 0px 0px 0px; 
 +
margin: 0px 0px 0px 0x;
 +
}
 +
h3
 +
{
 +
color:#262626;
 +
text-align:left;
 +
font-family:"Trebuchet";
 +
font-size:17px;
 +
line-height: 100%;
 +
border-bottom: none; 
 +
padding:0px 0px 0px 0px; 
 +
margin: 0px 0px 0px 0x;
 +
}
 +
p
 +
{
 +
color:#262626;
 +
text-align: justify;
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font-family:"Arial";
 +
font-size:14px;
 +
line-height:100%
 +
border-bottom: none;
 +
padding:0px 0px 0px 0px; 
 +
margin: 0px 0px 0px 0x;
 +
border:0px
 +
}
 +
body {
 +
color:#808080;
 +
line-height: 1;
 +
font-family:"Arial";
 +
font-size:11px; 
 +
}
 +
 +
.whitebox {
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  -moz-box-shadow:  0px 0px 0px #ccc;
 +
  -webkit-box-shadow:  0px 0px 0px #ccc;
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  box-shadow:  0px 0px 15px #ccc;
 +
  background-color: #FFFFFF;
 +
  -moz-border-radius: 1px;
 +
  border-radius: 15px;
 +
  margin-top: 20px;
 +
  padding-top: 20px;
 +
  padding-left: 20px;
 +
  padding-right: 20px;
 +
  padding-bottom: 20px;
 +
  margin-left: 15px;
 +
  margin-right: 25px;
 +
}
 +
.thumbnail
 +
{
 +
margin-bottom:0px;
 +
padding-bottom:0px;
 +
position:relative;
 +
}
 +
 +
#igemuwmenu, #igemuwmenu ul {
 +
margin: 0;
 +
padding: 0;
 +
list-style: none;
 +
}
 +
 +
#igemuwmenu {
 +
width: 820px;
 +
margin: 10px auto;
 +
border: 1px solid #222;
 +
background-color: #111;
 +
background-image: -moz-linear-gradient(#444, #111);
 +
background-image: -webkit-gradient(linear, left top, left bottom, from(#444), to(#111));
 +
background-image: -webkit-linear-gradient(#444, #111);
 +
background-image: -o-linear-gradient(#444, #111);
 +
background-image: -ms-linear-gradient(#444, #111);
 +
background-image: linear-gradient(#444, #111);
 +
-moz-border-radius: 6px;
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-webkit-border-radius: 6px;
 +
border-radius: 6px;
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-moz-box-shadow: 0 1px 1px #777, 0 1px 0 #666 inset;
 +
-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;
 +
                display: inline-block;
 +
                white-space: nowrap;
 +
}
 +
 +
#igemuwmenu:before,
 +
#igemuwmenu:after {
 +
content: "";
 +
display: table;
 +
}
 +
 +
#igemuwmenu:after {
 +
clear: both;
 +
}
 +
 +
#igemuwmenu {
 +
zoom:1;
 +
}
 +
 +
#igemuwmenu li {
 +
border-right: 1px solid #222;
 +
-moz-box-shadow: 1px 0 0 #444;
 +
-webkit-box-shadow: 1px 0 0 #444;
 +
box-shadow: 1px 0 0 #444;
 +
position: relative;
 +
                display: inline-block;
 +
                white-space: nowrap;
 +
}
 +
 +
#igemuwmenu a {
 +
padding: 12px 20px;
 +
color: #999;
 +
text-transform: uppercase;
 +
font: bold 11.4px Arial, Helvetica;
 +
text-decoration: none;
 +
text-shadow: 0 1px 0 #000;
 +
                display: inline-block;
 +
                white-block: nowrap;
 +
}
 +
       
 +
    #igemuwmenu li:hover > a {
 +
color: #fafafa;
 +
}
 +
 +
*html #igemuwmenu li a:hover { /* IE6 only */
 +
color: #fafafa;
 +
}
 +
 +
#igemuwmenu ul {
 +
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;
 +
position: absolute;
 +
top: 38px;
 +
left: 0;
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z-index: 1;   
 +
background: #444;
 +
background: -moz-linear-gradient(#444, #111);
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background-image: -webkit-gradient(linear, left top, left bottom, from(#444), to(#111));
 +
background: -webkit-linear-gradient(#444, #111);   
 +
background: -o-linear-gradient(#444, #111);
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background: -ms-linear-gradient(#444, #111);
 +
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 {
 +
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;
 +
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;
 +
box-shadow: 0 1px 0 #111, 0 2px 0 #666;
 +
}
 +
 +
 +
#igemuwmenu ul li:last-child { 
 +
-moz-box-shadow: none;
 +
-webkit-box-shadow: none;
 +
box-shadow: none;   
 +
}
 +
 +
#igemuwmenu ul a {   
 +
padding: 10px;
 +
width: 130px;
 +
_height: 10px; /*IE6 only*/
 +
display: block;
 +
white-space: nowrap;
 +
float: none;
 +
text-transform: none;
 +
}
 +
     
 +
        #igemuwmenu ul a.selected {   
 +
padding: 10px;
 +
width: 130px;
 +
_height: 10px; /*IE6 only*/
 +
display: block;
 +
white-space: nowrap;
 +
float: none;
 +
text-transform: none;
 +
}
 +
 +
#igemuwmenu ul a:hover {
 +
background-color: #785D84;
 +
background-image: -moz-linear-gradient(#B6A0BA,  #785D84);
 +
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);
 +
background-image: linear-gradient(#B6A0BA, #785D84);
 +
}
 +
 +
#igemuwmenu ul li:first-child > a {
 +
-moz-border-radius: 3px 3px 0 0;
 +
-webkit-border-radius: 3px 3px 0 0;
 +
border-radius: 3px 3px 0 0;
 +
}
 +
 +
#igemuwmenu ul li:first-child > a:after {
 +
content: '';
 +
position: absolute;
 +
left: 40px;
 +
top: -6px;
 +
border-left: 6px solid transparent;
 +
border-right: 6px solid transparent;
 +
border-bottom: 6px solid #444;
 +
}
 +
 +
#igemuwmenu ul ul li:first-child a:after {
 +
left: -6px;
 +
top: 50%;
 +
margin-top: -6px;
 +
border-left: 0;
 +
border-bottom: 6px solid transparent;
 +
border-top: 6px solid transparent;
 +
border-right: 6px solid #3b3b3b;
 +
}
 +
 +
#igemuwmenu ul li:first-child a:hover:after {
 +
border-bottom-color: #B6A0BA;
 +
}
 +
 +
#igemuwmenu ul ul li:first-child a:hover:after {
 +
border-right-color: #0299d3;
 +
border-bottom-color: transparent;
 +
}
 +
 +
#igemuwmenu ul li:last-child > a {
 +
-moz-border-radius: 0 0 3px 3px;
 +
-webkit-border-radius: 0 0 3px 3px;
 +
border-radius: 0 0 3px 3px;
 +
}
 +
 +
/* Mobile */
 +
#igemuwmenu-trigger {
 +
display: none;
 +
}
 +
 +
@media screen and (max-width: 600px) {
 +
 +
/* nav-wrap */
 +
#igemuwmenu-wrap {
 +
position: relative;
 +
}
 +
 +
#igemuwmenu-wrap * {
 +
-moz-box-sizing: border-box;
 +
-webkit-box-sizing: border-box;
 +
box-sizing: border-box;
 +
}
 +
 +
/* menu icon */
 +
#igemuwmenu-trigger {
 +
display: block; /* show menu icon */
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height: 40px;
 +
line-height: 40px;
 +
cursor: pointer;
 +
padding: 0 0 0 35px;
 +
border: 1px solid #222;
 +
color: #fafafa;
 +
font-weight: bold;
 +
background-color: #111;
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==''''''==
==''''''==
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<b>General Guidelines:</b>
<b>General Guidelines:</b>
<br>
<br>
-
<a href = "http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html/">PCR Primer Design Guidelines</a><br>
+
<a href = "http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html">PCR Primer Design Guidelines</a><br>
<br>
<br>

Latest revision as of 23:53, 14 September 2013


'

Primer Design

IDT’s Oligo Analyzer

Finnezymes Multiple primer analyzer
Tyler’s primer design video- Primer Design for PCR

General Guidelines:
PCR Primer Design Guidelines


Primer Design
Forward Primer:
Take the beginning of the 18~35 bases of your targeted sequence according to the melting temperature for the gene primer bind to the plasmid you want to transform into. Tms should be around 58*C to match VF2 and VR primers, unless making “hanging primers” by adding the prefix and suffix for cloning - these Tms can be a bit lower to make the combined oligo shorter.
Add the *prefix “gaattcgcggccgcttctagag” at the beginning (extend if needed)
Reverse primer:
Add the *suffix “tactagtagcggccgctgcag” to the end of the targeted sequence
Then reverse complement the whole thing (end of the 20 bases + suffix). Both primers must be listed 5’->3’ for ordering format purposes.

General notes:
-When finding the reverse complement of the end sequence, instead of writing out the reverse complement either use http://www.bioinformatics.org/sms/rev_comp.html or highlight the sequence in Ape, right click and click “Copy Rev-Com”
- The primer has to be at least 18 base pairs long ( NO LESS )
- The primer should end on an G/C for stability
When possible use the Oligo Analyzer or Multiple primer analyzer (ignore Tms):
- Click on hairpin, and looking at the structures the site pops out with. If there is a delta G value of <-2.0 add more base pairs till all values of any resulting structures are >-2.0. The reason for this is because of free energy, the primer might loop onto itself and not function anymore.
Using Ape:
- The highlighted region that you will use for primers will show “Tm” = mel temps. It should be between 40-50 degrees (THIS HAS MORE PRIORITY THAN THE delta G)

*the prefix and the suffix can be found in the following website:
http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix
(use the prefix for the non-inframe cloning, i.e. the longer one)
*match the melting temperature of forward and reverse primer
*A good way to check whether you design the primer correctly is used “find” (control +F) on a new plasmid file you construct by inserting the segment between the prefix and suffix.

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