Biomod/2013/CUMC/Methods.html: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 1: Line 1:
<html>
<html>
<head>
<head>
<meta charset="utf-8">
<meta charset="utf-8">
<meta name="viewport" content="width=device-width, initial-scale=1">
<meta name="viewport" content="width=device-width, initial-scale=1">
<title>Results and Discussion</title>
<title>Materials and Methods</title>
<!-- <link href="../lts_website/boilerplate.css" rel="stylesheet" type="text/css"> -->
<!-- <link href="../lts_website/boilerplate.css" rel="stylesheet" type="text/css"> -->
<!-- <link href="../lts_website/styles/responsive2.css" rel="stylesheet" type="text/css"> -->
<!-- <link href="../lts_website/styles/responsive2.css" rel="stylesheet" type="text/css"> -->
Line 92: Line 93:


<![endif]-->
<![endif]-->
</head>


</head>
<body>
<body>
<div class="gridContainer clearfix">
<div class="gridContainer clearfix">
<div id="div1" class="fluid clearfix">
<div id="div1" class="fluid clearfix">
  <table width="100%" border="0" cellpadding="0">
  <table width="100%" border="0" cellpadding="0">
Line 125: Line 125:
<li><a href='index2.html'>Home</a></li>  
<li><a href='index2.html'>Home</a></li>  
<li><a href='about.html'>About Us</a></li>  
<li><a href='about.html'>About Us</a></li>  
<li><a href='Results.html'>Project<font size='1'>&#9660;</font></a>  
<li><a href='Methods.html'>Project<font size='1'>&#9660;</font></a>  
<ul class='menus'>  
<ul class='menus'>  
<li><a href='Intro.html'>Abstract and Introduction</a></li>
<li><a href='Intro.html'>Abstract and Introduction</a></li>
<li><a href='Methods.html'>Materials and Methods</a></li>  
<li><a href='Methods.html'>Materials and Methods</a></li>  
<li><a href='Results.html'>Results and Discussion</a></li>  
<li><a href='Results.html'>Results and Discussion</a></li>
<li><a href='Conclusion.html'>Conclusion</a></li>  
<li><a href='Conclusion.html'>Conclusion</a></li>  
<li><a href='https://skydrive.live.com/view.aspx?resid=6006BCA28B2E2BAF!123&app=OneNote&authkey=!AC3_eA_y21JncjE'>Lab Notes</a></li>  
<li><a href='https://skydrive.live.com/view.aspx?resid=6006BCA28B2E2BAF!123&app=OneNote&authkey=!AC3_eA_y21JncjE'>Lab Notes</a></li>  
Line 136: Line 136:
</ul>  
</ul>  
</li>  
</li>  
<li><a href='Results.html'>Related Sites<font size='1'>&#9660;</font></a>  
<li><a href='Methods.html'>Related Sites<font size='1'>&#9660;</font></a>  
<ul class='menus'>  
<ul class='menus'>  
<li><a href='http://www.cumc.columbia.edu/dept/medicine/BasicResearchers/Stojanovic.html'>Stojanovic Research Site</a></li>  
<li><a href='http://www.cumc.columbia.edu/dept/medicine/BasicResearchers/Stojanovic.html'>Stojanovic Research Site</a></li>  
</ul>  
</ul>  
</li>   
</li>   
      </ul>  
    </ul>  
    </nav>
  </nav>
    </div>
</div>
     <table width="100%" border="0" height="10">
     <table width="100%" border="0" height="10">
   <tr>
   <tr>
Line 149: Line 149:
   </tr>
   </tr>
</table>
</table>
<div id="div3.1" class="clearfix">
<h1>Materials and Methods</h1>
<h2><span class="mw-headline" style="font-size: 20px">Materials</span></h2>
<p style="text-align: left; font-size: 16px;"> TRIS, EDTA, MgCl2, NaCl, CaCl were purchased from Fisher
Scientific. Single stranded m13mp18 DNA strands were purchased from New England
Biolabs. Single stranded oligo staples were designed through caDNAno and purchased
through Integrated DNA Technologies. All sequences can be found in the
supplementary section.</p>


<h1>Results and Discussion</h1>
<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Bundle Structure</span></h2>
<h2><span class="mw-headline" style="font-size: 20px">Structural Designs</span></h2>
<p style="font-size: 16px; text-align: left;"; text-align: left;">Folding solution consisted of 20% 5X salt
<p>All structural designs were done on caDNAno which is an open source plugin
solution of EDTA, TRIS, NaCl, and MgCl2. Solution was thermally annealed from 56 degrees C
developed at Wyss Institute for Autodesk Maya; each design, once completed, was then
to 44 degrees C at a rate of 0.2 degrees C/12 minutes. The structures were purified away from the
verified using a software tool called CanDo which is provided by Laboratory for
excess of short staple using a 100kDa MWCO Millipore Amicon centrifugal filter spun
Computational Biology & Biophysics at MIT . CanDo imports the raw data from
down three rounds of 15 minutes each using three 500?l volumes folding buffer solution.
caDNAno files and outputs a 3D model of the predicted DNA structure. These models
The purified bundle structures were subsequently mixed with the inner barrel structure
show the expected shape, capsule-like figure for the barrels, and the basket weave
for assembly. </p>
panel at the end of both barrels. The basket weave end has an intertwined section of
scaffold and staples (Figure 2).</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/CapsuleModel_zps6cd5ff24.png" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>
 
<h2><span class="mw-headline" style="font-size: 20px">Optimizing Folding Conditions</span></h2>
 
<p>Optimization of the origami structures found that each structure has unique
folding salt concentration as well as thermal annealing conditions. The bundle structure
folded under 1mM EDTA, 5mM TRIS, 5mM NaCl, and 20mM MgCl2 salt concentrations.
Thermal annealing conditions included 12 hour cycle starting at 56 degrees C and ending at 44
degrees C with a rate of -0.2 degrees C /12 minutes. TEM imaging verified the folding of this structure
(Figure 3).</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/TEM1_zps5aec1176.png" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>
<p>The inner barrel was run on a matrix of differing magnesium and calcium concentrations.
Optimal conditions included 0.5mM EDTA, 5mM TRIS, 2.5mM CaCl2, and 4.6mM MgCl2.
Yield of formation hit its peak at a 12 hour folding reaction starting at 71 degrees C and ending
at 44 degrees C with a rate of -0.3 degrees C / 8 minutes. Longer annealing time at this point did not
lower the yield, nor did it increase it. Gel results showed a strong dimer band (Figure 4).</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/Gel1_zps08739707.png" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>
 
<p>The monomer was verified as the properly formed structure (Figure 5). The dimer,
however, turned out to be two inner barrel structures non-specifically binding to each
other from the panel end, also known as blunt stacking (Figure 6). The basket weaved
back panel was also verified to be present in the structure due to the dark negative stain
in one end of the capsule, signifying the strong amount of DNA present in that single
section.</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/TEM2_zps39d14f93.png
" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>
 
<p>The outer barrel was initially tried under the same conditions as the inner barrel
due to the near identical structural design. However, folding salt concentrations differedslightly consisting of 0.5mM EDTA, 5mM TRIS and 2mM MgCl2. The thermal annealing
conditions were the same as the 12 hour annealing optimized for the inner barrel.</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/Fig6_zps3c3306a3.png
" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/Fig6Cap_zpscb61f523.png
" width="57%" height="49"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>


<h2><span class="mw-headline" style="font-size: 20px">Modifications to Assemble Capsule</span></h2>
<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Inner and Outer Barrel Structures</span></h2>
<p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> Folding solution consisted of 20%
5X salt solution of EDTA, TRIS, CaCl, and MgCl2. Solution was thermally annealed from
71 degrees C to 44 degrees C at a rate of 0.3 degrees C /8 minutes. The samples were likewise purified using a
100k Millipore filter spun down three rounds of 15 minutes each. The samples were
then mixed post-fold to assemble. </p>


<p>After verification of the
<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Post Fold Assembly</span></h2>
structures forming properly
<p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> After structures were mixed they were
under TEM images, as well as
heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2%
its stability in physiological
agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial
buffer, modifications were
nucleic acid purification kit.</p>
added to the inner barrel help
guide the energetically
disfavored assembly of these
structures. The inner barrel features a series of short 20 bp strands that extend toward
the interior of the structure. These staples line up the inner wall of the structure from the
lip all the way to the base. Each staple binds to a specific series of regions of the 400 bp
loop which extends from one end bundle structure. The staples bind to the loop in
successive order to pulling the bundle inside the inner barrel structure. The two
structures showed signs of assembly under a post-mix heating of a constant 37 degrees C for
one hour. In Figure 3 there is a clear contrast in stain from the body of the inner barrel
structure to the basket weave panel; this is due to the difference in the density of DNA
in those respective locations. Images of the bundle and barrel solution under with the
staple modifications for post-fold assembly show a high negative stain throughout the whole inner barrel structure. (Figure 7). This shows signs of success in the assembly
technique by the usage of hybridizing extended staples for 3D origami structures.</p>
 
<table width="44%" border="0" align="center">
  <tr>
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/TEM3_zps6c2ff55a.png
" width="100%"></a></td>
    <th scope="col">&nbsp;</th>
  </tr>
</table>


<p>Further investigation in the complete assembly should focus first on completing
<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Post Fold Assembly</span></h2>
the inner-outer barrel assembly. The 400 bp loop in the outer barrel is tightly held
<p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> After structures were mixed they were
against the lip of the barrel so that the extended staples of the inner barrel can find its
heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2%
hybridizing regions.</p>
agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial
nucleic acid purification kit.</p>


<table width="44%" border="0" align="center">
<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Gel Electrophoresis</span></h2>
  <tr>
<p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> 2% Agarose gels were used that consisted of MgCl2 and
  <td width="100%" align="center" valign="top"><a class="fancybox"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/Rendering_zpsa6beeae0.png
Ethidium Bromide for stain under UV imaging. Gels were run for two hours at 120V with
" width="100%"></a></td>
a circulating pump maintain a uniform 6°C buffer solution.</p>
    <th scope="col">&nbsp;</th>
  </tr>
</table>


<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">TEM Imaging</span></h2>
<p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> TEM images were taken with a negative stain. All purified
samples imaged using TEM imaging were diluted to a 4 nM concentration.</p>


<table width="100%" class="rounded" border="0" style="background-image:url(http://i920.photobucket.com/albums/ad48/himynameisparth/gray_gradient_zps8eb773b0.jpg); border-image:round;">
<table width="100%" class="rounded" border="0" style="background-image:url(../lts_website/images/gray_gradient.jpg); border-image:round;">
   <tr>
   <tr>
     <td rowspan="2"><table width="100%" border="0">
     <td rowspan="2"><table width="100%" border="0">
Line 282: Line 202:
         </tr>
         </tr>
     </table></td>
     </table></td>
     <td height="36" align="right" class="visit3">© 2013 by NanoMechanists</td>
     <td height="36" align="right" class="visit3">© 2013 by NanoMechanists</td>
   </tr>
   </tr>
   <tr>
   <tr>

Revision as of 02:13, 27 October 2013

<html>

<head> <meta charset="utf-8"> <meta name="viewport" content="width=device-width, initial-scale=1"> <title>Materials and Methods</title> <!-- <link href="../lts_website/boilerplate.css" rel="stylesheet" type="text/css"> --> <!-- <link href="../lts_website/styles/responsive2.css" rel="stylesheet" type="text/css"> --> <!-- <link href="../lts_website/styles/responsive_style.css" rel="stylesheet" type="text/css"> --> <!-- <link href="../lts_website/styles/menu5.css" rel="stylesheet" type="text/css"> --> <style> body { margin: 0px; }

  1. menu{

background-color:#0CF; color: #FFF; height: 40px; border-bottom: 2px solid #DDD; box-shadow: 1px 2px 9px #B1B1B1; border-top: 2px solid #DDD; }

  1. menu ul,#menu li{margin:0 auto;padding:0 0;list-style:none}
  2. menu ul{height:45px;width:100%;}
  3. menu li{float:left;display:inline;position:relative;font:bold 0.8em Arial;text-shadow: 1px 2px 4px #838383;}
  4. menu a{display: block;

line-height: 40px; padding: 0 14px; text-decoration: none; color: #FFF; }

  1. menu li a:hover{

color:#CCC; -webkit-transition: all .1s ease-in-out; -moz-transition: all .1s ease-in-out; -ms-transition: all .1s ease-in-out; -o-transition: all .1s ease-in-out; transition: all .1s ease-in-out; background:#666; }

  1. menu input{display:none;margin:0 0;padding:0 0;width:80px;height:30px;opacity:0;cursor:pointer}
  2. menu label{font:bold 30px Arial;display:none;width:35px;height:36px;line-height:36px;text-align:center}
  3. menu label span{font-size:16px;position:absolute;left:35px}
  4. menu ul.menus{

height: auto; overflow: hidden; width: 170px; background: #50B7DC; position: absolute; z-index: 99; display: none; }

  1. menu ul.menus li{

display: block; width: 100%; font:normal 0.8em Arial; text-transform: none; text-shadow: none; border-bottom: 1px dashed #31AFDB; }

  1. menu ul.menus a{

color: #FFF; line-height: 35px; }

  1. menu li:hover ul.menus{display:block}
  2. menu ul.menus a:hover{

background: #5FC6EB; color: #FFF; -webkit-transition: all .1s ease-in-out; -moz-transition: all .1s ease-in-out; -ms-transition: all .1s ease-in-out; -o-transition: all .1s ease-in-out; transition: all .1s ease-in-out; } @media screen and (max-width: 1050px){ 

 #menu{position:relative} 
 #menu ul{background:#111;position:absolute;top:100%;right:0;left:0;z-index:3;height:auto;display:none} 
 #menu ul.menus{width:100%;position:static;padding-left:20px} 
 #menu li{display:block;float:none;width:auto; font:normal 0.8em Arial;} 
 #menu input,#menu label{position:absolute;top:0;left:0;display:block} 
 #menu input{z-index:4} 
 #menu input:checked + label{color:white} 
 #menu input:checked ~ ul{display:block}

} </style> <!-- <script type="text/javascript" src="http://code.jquery.com/jquery-latest.min.js"></script> --> <!-- <link rel="stylesheet" href="../lts_website/_notes/source/jquery.fancybox.css" type="text/css" media="screen"> --> <!-- <script type="text/javascript" src="../lts_website/_notes/source/jquery.fancybox.pack.js"></script> -->



<![endif]--> </head>

<body> <div class="gridContainer clearfix"> <div id="div1" class="fluid clearfix"> <table width="100%" border="0" cellpadding="0"> <tr>

</tr> </table> 

     </div>

<div id="div2" class="fluid clearfix"> 

 <table width="100%" border="0" cellpadding="5">
   <tr>
     <td width="47%" rowspan="2" style="min-width:20px;"><a class="fancybox" href="http://www.cumc.columbia.edu/"><img src="http://i920.photobucket.com/albums/ad48/himynameisparth/columbia_logo_zpse5bce01f.jpg" width="215" height="100" align="top"></a></td>
     <td width="53%" height="53" align="right" valign="bottom" style="text-align: right"><p>Columbia University NanoMechanists</p>
       </td>
   </tr>
   
 </table>

</div>

<div id="div10" class="clearfix fluid">

<nav id='menu'> <input type='checkbox'/> <label>&#8801;<span>Navigation</span></label> <ul class="fluid"> <li><a href='index2.html'>Home</a></li> <li><a href='about.html'>About Us</a></li> <li><a href='Methods.html'>Project<font size='1'>&#9660;</font></a> <ul class='menus'> <li><a href='Intro.html'>Abstract and Introduction</a></li> <li><a href='Methods.html'>Materials and Methods</a></li> <li><a href='Results.html'>Results and Discussion</a></li> <li><a href='Conclusion.html'>Conclusion</a></li> <li><a href='https://skydrive.live.com/view.aspx?resid=6006BCA28B2E2BAF!123&app=OneNote&authkey=!AC3_eA_y21JncjE'>Lab Notes</a></li> <li><a href='Images.html'>Images</a></li> <li><a href='../lts_website/vid.html'>Video</a></li> </ul> </li> <li><a href='Methods.html'>Related Sites<font size='1'>&#9660;</font></a> <ul class='menus'> <li><a href='http://www.cumc.columbia.edu/dept/medicine/BasicResearchers/Stojanovic.html'>Stojanovic Research Site</a></li> </ul> </li> 

   </ul> 
 </nav>

</div> 

   <table width="100%" border="0" height="10">
 <tr>
   <td>&nbsp;</td>
 </tr>

</table> <div id="div3.1" class="clearfix"> <h1>Materials and Methods</h1> <h2><span class="mw-headline" style="font-size: 20px">Materials</span></h2> <p style="text-align: left; font-size: 16px;"> TRIS, EDTA, MgCl2, NaCl, CaCl were purchased from Fisher Scientific. Single stranded m13mp18 DNA strands were purchased from New England Biolabs. Single stranded oligo staples were designed through caDNAno and purchased through Integrated DNA Technologies. All sequences can be found in the supplementary section.</p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Bundle Structure</span></h2> <p style="font-size: 16px; text-align: left;"; text-align: left;">Folding solution consisted of 20% 5X salt solution of EDTA, TRIS, NaCl, and MgCl2. Solution was thermally annealed from 56 degrees C to 44 degrees C at a rate of 0.2 degrees C/12 minutes. The structures were purified away from the excess of short staple using a 100kDa MWCO Millipore Amicon centrifugal filter spun down three rounds of 15 minutes each using three 500?l volumes folding buffer solution. The purified bundle structures were subsequently mixed with the inner barrel structure for assembly. </p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Inner and Outer Barrel Structures</span></h2> <p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> Folding solution consisted of 20% 5X salt solution of EDTA, TRIS, CaCl, and MgCl2. Solution was thermally annealed from 71 degrees C to 44 degrees C at a rate of 0.3 degrees C /8 minutes. The samples were likewise purified using a 100k Millipore filter spun down three rounds of 15 minutes each. The samples were then mixed post-fold to assemble. </p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Post Fold Assembly</span></h2> <p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> After structures were mixed they were heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2% agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial nucleic acid purification kit.</p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Preparation of Post Fold Assembly</span></h2> <p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> After structures were mixed they were heated at a constant rate of 37 degrees C for one hour. These samples were run on a 2% agarose gel to verify the folding, and purified using “freeze & squeeze” a commercial nucleic acid purification kit.</p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">Gel Electrophoresis</span></h2> <p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> 2% Agarose gels were used that consisted of MgCl2 and Ethidium Bromide for stain under UV imaging. Gels were run for two hours at 120V with a circulating pump maintain a uniform 6°C buffer solution.</p>

<h2><span class="mw-headline" style="text-align: left; font-size: 20px;">TEM Imaging</span></h2> <p style="font-size: 16px; text-align: left; color: #009;"; text-align: left;"> TEM images were taken with a negative stain. All purified samples imaged using TEM imaging were diluted to a 4 nM concentration.</p>

<table width="100%" class="rounded" border="0" style="background-image:url(../lts_website/images/gray_gradient.jpg); border-image:round;"> 

 <tr>
   <td rowspan="2"><table width="100%" border="0">
     <tr>
       <td height="34" align="left" valign="top" class="visit2">Stojanovic Research Group</td>
       </tr>
   </table></td>
   <td height="36" align="right" class="visit3">©  2013 by NanoMechanists</td>
 </tr>
 <tr>
   <td align="right" valign="bottom" class="visit3">Site is best viewed in full screen (site adapts to screen size)<br></td>
 </tr>

</table> </body> </html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2013/CUMC/Methods.html&oldid=750797"