Biomod/2011/TUM/TNT/LabbookA/Labeling oligonucleotides with fluorophores

From OpenWetWare
Jump to: navigation, search


  1. column-one { display:none; width:0px;}

.container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}

  1. content { width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
  1. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. globalWrapper{ width:1280px;}

div.tright { border-bottom-width: 0em; border-left-width: 0px; border-right-width: 0px; border-top-width: 0em; clear: right; float: right;


div.tleft { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; clear: left; float: left; margin-right: 0.5em;


div.tnone { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; }

  1. column-one {display:none; width:0px;background-color: #ffffff;}
  1. content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f5f5f5; }

.container{ width: 800px; margin: auto; background-color: #ffffff; text-align:justify; font-family: helvetica, arial, sans-serif; color:#555555; margin-top:25px; }

  1. bodyContent{ width: 1267px; align: center; background-color: #ffffff;}
  1. column-content{width: 1280px;background-color: #f5f5f5;}

.firstHeading { display:none;width:0px;background-color: #ffffff;}

  1. header{position: center; width: 800px;background-color: #ffffff;}
  1. footer{position: center;}

<style type="text/css"> body {

   font-family: helvetica, arial, sans-serif;
   color: black;


ul#topnav {

margin: 0 0 0 0px ; padding: 0 0 0 0px; float: left; width: 800px; list-style: none; position: relative;

       font-family: helvetica, arial, sans-serif;

font-size: 12px; background: #f5f5f5;

       height: 5px;

} ul#topnav li { float: left; margin: 0; padding: 0; border-right: 1px solid #ffffff; /*--Divider for each parent level links--*/ } ul#topnav li a { padding: 11px 15px; display: block; color: #333333; text-decoration: none; } ul#topnav li:hover { background: #f5f5f5;} /*--Notice the hover color is on the list item itself, not on the link. This is so it can stay highlighted even when hovering over the subnav--*/

ul#topnav li span { float: left; padding: 15px 0; position: absolute; left: 0; top:30px; display: inline; /*--Hide by default--*/ width: 800px; background: #cccccc; color: #333333; /*--Bottom right rounded corner--*/ -moz-border-radius-bottomright: 5px; -khtml-border-radius-bottomright: 5px; -webkit-border-bottom-right-radius: 5px; /*--Bottom left rounded corner--*/ -moz-border-radius-bottomleft: 5px; -khtml-border-radius-bottomleft: 5px; -webkit-border-bottom-left-radius: 5px; } ul#topnav li:hover span { display: block; } /*--Show subnav on hover--*/ ul#topnav li span a { display: inline;} /*--Since we declared a link style on the parent list link, we will correct it back to its original state--*/ ul#topnav li span a:hover {text-decoration: underline;}

  1. abstractandlinks{

display:block; width:800px; background-color:#f5f5f5; text-align:justify;font-family: helvetica, arial, sans-serif; color:#555555; margin-top:0px; }

  1. innerBox

{ display: block; padding: 5px; }

  1. abstract{

font-family: helvetica, arial, sans-serif; width: 490px; margin-top: 20px; padding:0px 5px; float: left;background-color:#f5f5f5; ; }

  1. links{

font-family: helvetica, arial, sans-serif; width: 290px; margin-top: 20px; padding:0px 5px; float: left; background-color:#f5f5f5;; } .clear { clear:both; }

  1. tour{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; margin-top: 1em, margin-bottom: 1em; float: left;background:#f5f5f5; font-size: 2em; }

  1. struktur{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; float: left;background:#f5f5f5; }

  1. toctitle {align: left; text-align: left; }
  2. table.toc {background-color:"#5f5f5f";align: left; text-align: left;}
  3. toc #toctitle, .toc #toctitle, #toc .toctitle, .toc .toctitle {

text-align: left;

  1. toc, .toc, .mw-warning {

background-color: #f5f5f5; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 0px; border-left-color: rgb(170, 170, 170); border-left-style: solid; border-left-width: 0px; border-right-color: rgb(170, 170, 170); border-right-style: solid; border-right-width: 0px; border-top-color: rgb(170, 170, 170); border-top-style: solid; border-top-width: 0px; font-size: 105%; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px; align: left; text-align: left; display: left




<script type="text/javascript">

$(document).ready(function() {

$("ul#topnav li").hover(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

$("ul#topnav li").oneclick(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'; 'display' : 'inline'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

function() { //on hover out...

$(this).css({ 'background' : 'none'}); //Ditch the background

//$(this).find("span").hide(); //Hide the subnav });

}); </script>


       TUM NanU - Home   

</title> </head>

<body background-color="#F0F0F0"> <!-- Header--> <div class="container">

   <div id="header"> <img src="" /> </div> 

<!--Menue--> <ul id="topnav">

   <li><a href="">Home</a></li>
   <li><a href="">Project</a>        
   <li><a href="">Results & Discussion</a>        
   <li><a href="">Methods</a></li>
   <li><a href="">Team</a></li>
<li style="background-color: #cccccc"><a href=""><b>Labbook</b></a><!--Subnav Starts Here-->
           <a href="">Main Page</a>||
           <a href="">Structure</a>|
           <a href="">TEM</a>|
           <a href="">Fluorescence Microscopy</a>|
           <a href="">Glossary</a>
   <li><a href="">Extras</a>

</ul> <p> <br> </p> <p> <br> </p> <p> <br> </p> </html>


<html> <head> <script type= "text/javascript">/*<![CDATA[*/ var skin = "monobook"; var stylepath = "/skins"; var wgArticlePath = "/wiki/$1"; var wgScriptPath = ""; var wgScript = "/index.php"; var wgVariantArticlePath = false; var wgActionPaths = []; var wgServer = ""; var wgCanonicalNamespace = ""; var wgCanonicalSpecialPageName = false; var wgNamespaceNumber = 0; var wgPageName = "Biomod/2011/TUM/TNT/LabbookA/2011"; var wgTitle = "Biomod/2011/TUM/TNT/LabbookA/2011 var wgAction = "view"; var wgArticleId = "103708"; var wgIsArticle = true; var wgUserName = "Alexander Hipp"; var wgUserGroups = ["*", "user", "autoconfirmed"]; var wgUserLanguage = "en"; var wgContentLanguage = "en"; var wgBreakFrames = false; var wgCurRevisionId = "543703"; var wgVersion = "1.13.2"; var wgEnableAPI = true; var wgEnableWriteAPI = false; var wgMWSuggestTemplate = "\x26search={searchTerms}\x26namespace={namespaces}"; var wgDBname = "owwdb"; var wgSearchNamespaces = [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 100, 101, 110, 111]; var wgMWSuggestMessages = ["with suggestions", "no suggestions"]; var wgRestrictionEdit = []; var wgRestrictionMove = []; var wgAjaxWatch = {"watchMsg": "Watch", "unwatchMsg": "Unwatch", "watchingMsg": "Watching...", "unwatchingMsg": "Unwatching..."}; /*]]>*/</script>

<script type="text/javascript" src="/skins/common/wikibits.js?164"><!-- wikibits js --></script> <!-- Head Scripts --> <script type="text/javascript" src="/js/calendar.js"></script> <script type="text/javascript"> function lnCalendar(){

 var calm1 = new CalendarPopup('m1');
 var wikiPage = 'Biomod/2011/TUM/TNT/LabbookA';
 var redirPage = '/wiki/Special:Redir/'+wikiPage;
 var fullDates = new Array('08/11/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');
 for(i = 0; i < fullDates.length; i++)calm1.addFilledDates(fullDates[i]);
 var calm2 = new CalendarPopup('m2');


 for(i = 0; i < fullDates.length; i++)calm2.addFilledDates(fullDates[i]);
 var calm3 = new CalendarPopup('m3');


 for(i = 0; i < fullDates.length; i++)calm3.addFilledDates(fullDates[i]);

} </script> <script type="text/javascript">addOnloadHook( lnCalendar )</script> <script type="text/javascript">function CalendarPageConfirmCreate(date, url){var answer = confirm("Create entry for "+date+"?");if (answer){window.location = url;}}</script> <script type="text/javascript" src="/skins/common/ajax.js?164"></script> <script type="text/javascript" src="/ext/AjaxShowEditors/AjaxShowEditors.js"></script> <script type="text/javascript" src="/skins/common/ajaxwatch.js?164"></script> <script type="text/javascript" src="/skins/common/mwsuggest.js?164"></script> <script type="text/javascript" src="/index.php?title=-&amp;action=raw&amp;smaxage=0&amp;gen=js&amp;useskin=monobook"><!-- site js --></script>

<style type="text/css">/*<![CDATA[*/ @import "/index.php?title=MediaWiki:Common.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=MediaWiki:Monobook.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=-&amp;action=raw&amp;gen=css&amp;maxage=18000&amp;smaxage=0&amp;ts=20111012100110&amp;useskin=monobook"; @import "/index.php?title=User:Alexander_Hipp/monobook.css&amp;action=raw&amp;ctype=text/css"; /*]]>*/</style> <script type="text/javascript" src="/index.php?title=User:Alexander_Hipp/monobook.js&amp;action=raw&amp;ctype=text/javascript"></script> </head>


Labeling oligonucleotides with fluorophores


We use always the same two fluorophors, Atto 550 and Atto 647N, for our FRET measurements. However, we need them attached to different staples for our FRET measurements. Therefore, we bought fluorophore labeled nucleotides, Atto 550 ddCTP and Atto 647N ddUTP. Using terminal tranferase, these labeled nucleotides can ba added at the 3' end of a freely chosen oligonucleotide.



  • 16µl 5x TdT reaction buffer (supplied with terminal tranferase)
  • 16µl 25mM CoCl2
  • 4µl 100µM oligonucleotide that should be labeled
  • 2nmol fluorophor labeled nucleotide, i.e. 2µl 1mM Atto 550 ddCTP or 4µl 0.5mM Atto 647N ddUTP
  • 2µl terminal transferase
  • add ddH2O to a final volume of 80µl

incubate at 37°C for 40 minutes
add 8µl 0.2mM EDTA (pH8)and incubate at 70°C for 10 minutes in order to stop reaction

For purification, the oligo is precipitated in ethanol and then washed several times.

  • add 25µl 3M NaOAc and 275µl 99+% EtOH
  • incubate on dry ice for 30 min
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and add 400µl 75% EtOH in water
  • vortex thoroughly
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and add 400 µl 75% EtOH in water
  • vortex thoroughly
  • centrifuge for 30 min at 4°C at 16100 rcf
  • carefully remove the supernatant and dissolve the pellet in 50µl 0.5x TBE 11mM MgCl2
  • incubate over night at 4°C until the oligonucleotide has completely dissolved
  • hide sample frim light to prevent photobleaching