Biomod/2011/TUM/TNT/Methods: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 1: Line 1:
{{TUMNanUHeaderMethods}}
{{TUMNanUHeaderMethods}}
<div id="abstractandlinks">
<div id="abstractandlinks">
<br>
<font size="6">Methods</font size="6">
<font size="6">Methods</font size="6">
<h1> Folding </h1>
<h1> Folding </h1>


Folding of an origami is a critical step. Besides the question whether a designed structure folds at all, the yield and the fraction of misfolded structures are also important. The yield determines which concentrations could be applied for further experiments. If significant amounts of misfoldings occur, thorough purification is needed, which in turn corresponds to reduced yield due to purification losses. For these reasons, it was not only aimed to design a structure which can be expected to fold easily, but some effort was also dedicated to testing different thermal folding ramps and check the efficiency of folding. <br>
Folding of an origami is a critical step. Besides the question whether a designed structure folds at all, the yield and the fraction of misfolded structures are also important. The yield determines which concentrations could be applied for further experiments. If significant amounts of misfoldings occur, thorough purification is needed, which in turn corresponds to reduced yield due to purification losses. For these reasons, it was not only aimed to design a structure which can be expected to fold easily, but some effort was also dedicated to testing different thermal folding ramps and check the efficiency of folding. <br>


<h2>Folding procedure</h2>
<h2>Folding Procedure</h2>
For more detailed information, please consult the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions instruction] in the labbook.  
For more detailed information, please consult the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions instruction] in the labbook.  
<h3>Prestocks</h3>
<h3>Prestocks</h3>
Several hundreds of staples have to be mixed for a folding batch. Those staples, which are frequently used together for different variants of the structure, were mixed in prestocks. Later on, for small modifications of the structure, only the appropriate prestock needs to be remixed, while all other parts could be used again. Prestocks may vary in the number of included staples, but in one prestock, all staples are equally concentrated (usually 10µl 100µM staple are mixed).  
Several hundreds of staples have to be mixed for a folding batch. Those staples, which are frequently used together for different variants of the structure, were mixed in prestocks. Later on, for small modifications of the structure, only the appropriate prestock needs to be remixed, while all other parts could be used again. Prestocks may vary in the number of included staples, but in one prestock, all staples are equally concentrated (usually 10µl 100µM staple are mixed).  


<h3>Working stocks</h3>
<h3>Working Stocks</h3>
For every structure variant, one working stock is mixed. Here, all needed prestocks are mixed in such a way, that all staples finally have the same concentration, 500nM.  
For every structure variant, one working stock is mixed. Here, all needed prestocks are mixed in such a way, that all staples finally have the same concentration, 500nM.  


<h3>Folding batch</h3>
<h3>Folding Batch</h3>
The folding batch contains [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary#F FOB20], 20nM scaffold and 200nM of each staple. Dye-labeled oligonucleotides are added extra at this step.  
The folding batch contains [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary#F FOB20], 20nM scaffold and 200nM of each staple. Dye-labeled oligonucleotides are added extra at this step.  


<h3>Thermal ramp</h3>
<h3>Thermal Ramp</h3>
Three different ramps were tested, which proceed at different velocities during the critical steps of folding. <br>
Three different ramps were tested, which proceed at different velocities during the critical steps of folding. <br>
<b>15_65: </b>
<b>15_65: </b>
Line 31: Line 26:
For this longest ramp, again the first part is cooling down from 65°C to 57°C by -1°C per 15 minutes. Then, the batch is cooled down by 1°C per 3 hours. Finally, temperature is set to 4°C.<br>
For this longest ramp, again the first part is cooling down from 65°C to 57°C by -1°C per 15 minutes. Then, the batch is cooled down by 1°C per 3 hours. Finally, temperature is set to 4°C.<br>


<h2>Validation of folding efficiency</h2>
<h2>Validation of Folding Efficiency</h2>
Success of folding was evaluated with agarose gel electrophoresis. Thereby, it could be investigated, whether additional bands besides the main structure do occur. Subsequently, these bands could be analyzed at the TEM. <br>
Success of folding was evaluated with agarose gel electrophoresis. Thereby, it could be investigated, whether additional bands besides the main structure do occur. Subsequently, these bands could be analyzed at the TEM. <br>


Line 53: Line 48:




<h1>Fluorescence microscopy</h1>
<h1>Fluorescence Microscopy</h1>


<h2>Principle of FRET</h2>
<h2>Principle of FRET</h2>
Line 79: Line 74:
where <math>F</math> <math>'_{\rm D}</math> and <math>F _{\rm D}</math> are the donor fluorescence intensities with and without an acceptor, respectively.
where <math>F</math> <math>'_{\rm D}</math> and <math>F _{\rm D}</math> are the donor fluorescence intensities with and without an acceptor, respectively.


<h2>Bulk measurements</h2>
<h2>Bulk Measurements</h2>
Some test bulk measurements were performed both with a photospectrometer and with a RT-PCR. Therefore we used the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 MH_255/256 18bp double helix] with FRET labels at both ends. Thus the distance of the labels should match the Förster distance. Excitation and emission spectra at the photospectrometer were used to determine optimal wavelengths for FRET measurements. At the more sensitive RT-PCR, Atto 550 was excited at 545 nm and fluorescence detected at 568 nm. Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the RT PCR's optical filters.  
Some test bulk measurements were performed both with a photospectrometer and with a RT-PCR. Therefore we used the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 MH_255/256 18bp double helix] with FRET labels at both ends. Thus the distance of the labels should match the Förster distance. Excitation and emission spectra at the photospectrometer were used to determine optimal wavelengths for FRET measurements. At the more sensitive RT-PCR, Atto 550 was excited at 545 nm and fluorescence detected at 568 nm. Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the RT PCR's optical filters.  


<h2>Single molecule measurements</h2>
<h2>Single Molecule Measurements</h2>


<h3>Immobilization</h3>
<h3>Immobilization</h3>
Line 97: Line 92:
[[Image:TIRF DSC4455.JPG | 600px|center | thumb|Fig. 1 used homemade fluorescence microscope at Dietz Lab]]<br>
[[Image:TIRF DSC4455.JPG | 600px|center | thumb|Fig. 1 used homemade fluorescence microscope at Dietz Lab]]<br>


<h2>Evaluation of data</h2>
<h2>Evaluation of Data</h2>
In order to calculate the extent of structure deformation represented by the chance in distance of the two flouropores we evaluated the acquired data in two different ways. On the one side we determined the FRET efficiency by analyzing a recorded video on the other we simply measured the distance of two spots by comparing the two pictured we received by alternating excitation.
In order to calculate the extent of structure deformation represented by the chance in distance of the two flouropores we evaluated the acquired data in two different ways. On the one side we determined the FRET efficiency by analyzing a recorded video on the other we simply measured the distance of two spots by comparing the two pictured we received by alternating excitation.
<br>
<br>

Revision as of 03:05, 2 November 2011

<html> 

  <style>
  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.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;}


div.thumb { border-bottom-color: rgb(255, 255, 255); border-bottom-style: solid; border-left-color: rgb(255, 255, 255); border-left-style: solid; border-right-color: rgb(255, 255, 255); border-right-style: solid; border-top-color: rgb(255, 255, 255); border-top-style: solid; margin-bottom: 0.5em; width: auto; 

}

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

}

body { 

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

background:#f5f5f5; 

 }

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 

}


</style>

<head>

<script type="text/javascript"> var fullDates = new Array('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','08/31/2011','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');

$(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>


<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.3/jquery.min.js"></script>


<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> 

 <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script>
 <script type="text/javascript">
  $(document).ready(function(){
   $('#lside1').mouseover(function() {
    $('#lside1').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside1').mouseleave(function() {
    $('#lside1').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
   $('#lside2').mouseover(function() {
    $('#lside2').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside2').mouseleave(function() {
    $('#lside2').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
   $('#lside3').mouseover(function() {
    $('#lside3').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside3').mouseleave(function() {
    $('#lside3').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
  });
 </script>

<style type="text/css"> 

  #side1 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside1 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

  #side2 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside2 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

  #side3 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside3 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

 </style>


<title> 

       TUM NanU - Home

</title> </head>

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

   <div id="header"> <img src="http://openwetware.org/images/5/5c/Header2.png" /> </div>

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

   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Home">Home</a></li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project">Project</a>        
    </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results">Results & Discussion</a>          
   </li>
   <li style="background-color: #cccccc"><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods"><b>Methods</b></a>
       <span>
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Folding">Folding</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Purification">Purification</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Labeling">Labeling</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#TEM">TEM</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Fluorescence_Microscopy">Fluorescence Microscopy</a>
       </span>
 </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Team">Team</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA">Labbook</a><!--Subnav Starts Here--> 


   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras">Extras</a>
       
  </li>

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

</body> </html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TUM/TNT/Methods&oldid=560504"