Tianjin protocol: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
(26 intermediate revisions by the same user not shown)
Line 24: Line 24:
         margin-right:0px;
         margin-right:0px;
width:1000px;
width:1000px;
height:150px;
height:350px;
z-index:1;
z-index:1;
        box-shadow: 0px 5px 8px rgba(0,0,0,0.4);
overflow: visible;
overflow: visible;
text-align: left;
text-align: left;
background-image: url(http://openwetware.org/images/9/9f/Wiki-top4.jpg);
background-image: url(http://openwetware.org/images/3/37/Header_tianjin.png);
         }
         }


Line 40: Line 41:
height:40px;
height:40px;
z-index:2;
z-index:2;
font-family: "Arial Black", Gadget, sans-serif;
font-family: Georgia, "Times New Roman", Times, serif;
text-align: left;
text-align: left;
background-image: url(http://openwetware.org/images/thumb/7/76/Tianjin_index02.jpg/800px-Tianjin_index02.jpg);
background-image: url(http://openwetware.org/images/thumb/7/76/Tianjin_index02.jpg/800px-Tianjin_index02.jpg);
Line 77: Line 78:
margin-bottom:40px;
margin-bottom:40px;
         z-index:60;
         z-index:60;
         font-family: "Arial Black", Gadget, sans-serif;
         font-family: Georgia, "Times New Roman", Times, serif;
         text-align: left;
         text-align: left;
}
}
Line 83: Line 84:
#navDiv > ul > li {
#navDiv > ul > li {
     float: left;
     float: left;
     margin-left: 60px;
     margin-left: 30px;
     position: relative;
     position: relative;
}
}
Line 295: Line 296:
         display:block;
         display:block;
         left:0px;
         left:0px;
width:980px;
width:961px;
z-index:3;
z-index:3;
        box-shadow: 0px 5px 8px rgba(0,0,0,0.4);
text-align: left;
text-align: left;
font-family: "Comic Sans MS", cursive;
font-family: Georgia, "Times New Roman", Times, serif;
font-size: 18px;
font-size: 18px;
color: #000;
color: #000;
background-color: #D2E9FF;
background-color: #ffffff;
         padding: 20px;
         padding: 20px;
         }
         }


/*hidden section*/
.firstHeading{display:none;}
#sidebar-main{display:none;}
#p-cactions{display:none;}
#p-personal{display:none;}


</style>
</style>
Line 314: Line 322:
<!--header-->
<!--header-->
<div id="apDiv1"></div>
<div id="apDiv1"></div>


<!--navigator starts here-->
<!--navigator starts here-->
Line 330: Line 339:
             <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
             <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
           <ul>
           <ul>
               <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#strand replacement reaction"><span>Strand Replacement Reaction</span></a></li>
               <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Three-arm locker"><span>Three-arm Locker</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#synthesis of au-dna-cy3"><span> Synthesis of AU-DNA-CY3</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Gold nanoparticle based photosensor"><span> Gold Nanoparticle Based Photosensor</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
                     <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
                 </ul>
                 </ul>
Line 337: Line 346:
        
        
             <li class='active'><a href="http://openwetware.org/wiki/Tianjin_protocol"><span>Protocol</span></a>  
             <li class='active'><a href="http://openwetware.org/wiki/Tianjin_protocol"><span>Protocol</span></a>  
    </li>
            <li class='active'><a href="http://openwetware.org/wiki/Biomod/2014/future.html"><span>Future Work</span></a>
    </li>
    </li>
            
            
Line 344: Line 356:
</ul>
</ul>
</div>
</div>


<div id="toolBackTo" class="back-to" style="left: 1175px; ">
<div id="toolBackTo" class="back-to" style="left: 1175px; ">
Line 354: Line 367:
<div id="apDiv10">
<div id="apDiv10">
</html>
</html>
== CY3 NHS ester labelingt:==
==Buffer Preparation==
(1).Calculate required amount of NHS ester:
(1)5×TBE: Add 54g Tris Base, 3.7224g EDTA Na<sub>2</sub>﹒2H<sub>2</sub>O  and 27.5g  borieacid in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL. Make the pH into 8.25～8.30.<br>
 
(2)1×TBE: Add 200mL of 5×TBE in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.<br>
NHS ester weight [mg] = 8 × amino compound weight [mg] × NHS ester molar weight [Da] / amino compound molar weight [Da].
(3)50×TAE: Add 242g Tris Base, 23g EDTA and 57.1mL  A cetic acid glacial  in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.<br>
 
(4)1×TAE: Add 20mL of 50×TAE in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.<br>
In our experiments, in order to label 100ug of amino-modified DNA (molar weight 10927 Dalton) with Cy3 NHS ester (molar weight 590.15Dalton), and obtain maximum yield of mono-labeled product, we use
 
8× 100ug × 590.15 Da /10297 Da = 45.85 ug of Cy3 dye NHS ester.
 
(2).Determine volume of reaction mixture: use 10ul volume which concentration is 5ug of amino-modified DNA per uL of mixture.
(3).Dissolve 1mg CY3 NHS ester in 22ul of DMF. After the reaction, NHS ester can be stored in solution for 1-2 months at -20ºC.
 
(4).Dissolve amino-modified DNA in 9ul of 0.1 M Tris buffer with pH 8.3-8.5. Note pH is the most important thing.  
 
(5).Add 1ul CY3 NHS ester solution to the solution of DNA, and vortex well. Keep the mixture at room temperature during at least 4 hours.
(6).Purify the conjugate using ethanol precipitation.
 
 
== Synthesis of AU-DNA-CY3:==
(1).Prepare 10 mM TCEP
 
(2).Prepare 13 nM gold nanoparticles: two kinds of gold nanoparticles with diameter of 13nm and 40nm
 
(3).Pipette 9μl of 1 mM DNA into a microcentrifuge tube
 
(4).Add 1μl of 500 mM acetate buffer (pH 5.2) and 1.5μl of 10 mM TCEP to the tube to activate the thiol-modified DNA. Incubate the sample at room temperature for 1 h.
 
(5).Transfer 3 ml of the already prepared gold nanoparticles （13 nM）to the centrifuge tube, and then add the TCEP-treated thiol DNA with gentle shaking by hand.
 
(6).Cap the centrifuge tube and store them in a drawer at room temperature for at least 16 h.
 
(7).Add 30 μl of 500 mM Tris acetate (pH 8.2) buffer drop wise to each tube with gentle hand shaking. The final Tris acetate concentration is 5 mM.
 
(8).Add 300 μl of 1 M NaCl dropwise to each tube with gentle hand shaking. Store the tube in a drawer for at least another day before use.
(9).Transfer 500 μl of functionalized particles into 1.5-ml microcentrifuge tubes, respectively.
 
(10).Centrifuge the two tubes at 16,110g at room temperature (23–25 °C) on a benchtop centrifuge for 15 min.
 
 
== Autocatalytic reaction system:==
(1).Harpin reaction: total volume 10ul
<html>
<body>
<table width="200" border="1" cellspacing="2" cellpadding="1" background-color: #99CCFF>
  <tr>
    <th scope="row"><span class="ys01">Water</span></th>
    <td><span class="ys01">7μL</span></td>
  </tr>
  <tr>
    <th scope="row"><span class="ys01">10×buffer</span></th>
    <td><span class="ys01">1μL</span></td>
  </tr>
  <tr>
    <th scope="row"><span class="ys01">10μMDNA</span></th>
    <td><span class="ys01"> 2μL</span></td>
  </tr>
</table>
</body>
</html>


Reaction condition:
==Gel Pouring==


(a)heating at 90°C for 5 minutes.
===PAGE Gel(10%)===


(b)Cooling on ice for 1 minute.
<table width="200" border="1" cellspacing="2" cellpadding="1">
 
(c)At room temperature for 30 minutes before use.
 
The hairpins A B were prepared in reaction buffer as follows:
 
10×buffer:
<table width="110" border="1" cellspacing="2" cellpadding="1" background-color: #99CCFF>
  <tr>
    <th scope="col">Material</th>
    <th scope="col">Concentration(mM) </th>
    <th scope="col">MW(g/mol)</th>
    <th scope="col"> Mass(g)</th>
  </tr>
  <tr>
    <th scope="row">MgCl<sub>2</sub>·6H<sub>2</sub>O</th>
    <td>4</td>
    <td>203</td>
    <td>0.8132</td>
  </tr>
  <tr>
    <th scope="row">KCl</th>
    <td>15</td>
    <td>74.55</td>
    <td>1.11825</td>
  </tr>
  <tr>
    <th scope="row">Tris-HCl</th>
    <td>10</td>
    <td>121.14</td>
    <td>1.2114</td>
  </tr>
</table>       
 
(2).reaction for A and I:
 
Mix 2μL A(at 2μM) and 2μL I(at 2μM), heating at 95°C for 5 minutes followed by cooling to room temperature over approximately 2.5 hours.
 
(3).reaction for B+(AI)：
Mix 2μl B (at 2μM) and 2μl (AI) to react at room temperature for 15 minutes.
 
== PAGE Gel:==
<table width="110" border="1" cellspacing="2" cellpadding="1">
   <tr>
   <tr>
     <th scope="col">Content</th>
     <th scope="col">Content</th>
Line 469: Line 383:
   </tr>
   </tr>
   <tr>
   <tr>
     <th scope="row">Page Gel 30% </th>
     <th scope="row">Acrylamide Gel 30% </th>
     <td>15ml</td>
     <td>15ml</td>
   </tr>
   </tr>
Line 490: Line 404:
   <tr>
   <tr>
     <th scope="row">Total</th>
     <th scope="row">Total</th>
     <td>30ml</td>
     <td>15ml</td>
   </tr>
   </tr>
</table>
</table>


prepare gel according the volumes in the table above
Prepare gel according the volumes in the table above<br>
Pour gel into casting tray, and insert comb (make sure there are no bubbles)<br>
Put the whole setup in 37℃ incubator for 30 min<br>
Fill in TBE-Buffer<br>
Prepare the sample by adding 6×loading buffer containing Golden View
 
===Agarose Gel===
 
1.Place 1.0(or 2.0) g of agarose in a clean 600 mL beaker on a scale.<br>
2.Prepare the 1×TAE buffer as described. <br>
3.Add 100 mL of 1×TAE buffer.<br>
4.Microwave for 2 minutes. <br>
5.If the solution is not clear, swirl and microwave for another 15 seconds. <br>
6.Cool the beaker in an ice water bath. Keep swirling the beaker to prevent polymerization. Add the thermometer and keep checking the temperature. <br>
7.When the solution reaches 60°C, remove it from the ice water bath. <br>
8.Add 5µL of EB. <br>
9.Pour the contents of the beaker into the gel cassette and insert the comb.<br>
10.Use a glass Pasteur pipet to push any bubbles to the corners of the gel. <br>
11.Leave the cassette covered and let the assembly sit for thirty minutes to complete ensure complete polymerization.<br>
 
==Origimi==
 
===Design===


Pour gel into casting tray, and insert comb (make sure there are no bubbles)
We want to design a two-dimensional DNA origami plate which can be folded by our locker into a roll. The dimensions of the origami plate are properly selected so the drug stays encapsulated inside it. The final dimensions are the following:<br>
Length: 50 nm<br>
Width: 35 nm<br>
Thickness: 2 nm<br>
To enable the origami to fold into a roll, the three arm locker should be combined with it. The staple strands in two corners of the origami plate are extended with the sequences of strand A, and the staple strands in the other two corners are extended with the sequences of strand C.<br>
We used the cadnano 2 program for our design.<br>
The scaffold used in this design is M13mp18 single strand DNA with 7249 nt, from New England Biolabs LTD. The desired structure and dimensions are achieved with the help of two types of staples:<br>
core: 58 staples give stability to the whole structure.<br>
edge: 4 staples give stability to the edges and with extending sequences of A or C.<br>


Put the whole setup in 37℃ incubator for 30 min
===Folding and Characterization===


Fill in TBE-Buffer
The mixture of scaffold and different staples were subjected to a thermal annealing process composed of 3 steps that allowed the folding. Different initial temperatures varying from 50.0 to 65.0 in step 2 were tested. Electrophoresis showed that low initial temperatures in step 2 have relatively high origami yield.<br>


Prepare the sample by adding 6× loading buffer containing Golden View
====Different Thermal Annealing Processes of DNA Origami====
[[Image:PCR0.png]]


Run the gel by 200 V and 30 min
==Formation of the Hairpins==


== Agarose Gel:==
A, B and C did a snap-cooling procedure to form hairpin structure：
Measure 3g for 3% gel agarose in 250mL flask.
heating at 90 ◦C for 5 minutes and cooling on ice for 1 minute. The hairpins were allowed to equilibrate at room temperature for 30 minutes before use.
System including: 60µL ddH2O, 30µL 10µM DNA(A,B,C),10µL 10×Reaction Buffer(RB) total volume is 100µL/tube. (3µM reaction)
For 6µM I:  30µL ddH2O, 60µL 10µM DNA,10µL 10×Reaction buffer


Add 100 mL 1X TBE into flask.
==Assembly of the Three-arm Locker==


Microwave for about 3 minutes to disolve the powder.
===Dilution oligonucleotides===


Gently swirl in an ice water bath until it is not hot for your hand
Add water to 100µM. Then 10µL 100µM DNA+90µL water are mixed to get 10µM DNA.


Add EB stain in the gel with concentration of 5ul per 100ml
===Make A, B and C form hairpins as described.===


Pour gel into casting tray, and insert comb
Make A, B and C form hairpins as described.


cast the gel and cool the whole setup in room temperature
===Assembly(conc:750nM)===


Prepare the sample by adding 6× loading buffer containing Golden View
(1)single:7.5μL hairpin A+22.5μL 1xRB, total vol 30μL<br>
(2)assemble: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 6μM I 7.5μL, total vol 30μL<br>
(3)leakage: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 7.5μL 1xRB, total vol 30μL<br>
(4)annealed: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 7.5μL 1xRB (95◦C for 5 minutes first) , total vol 30μL<br>
(5)(ABCI<sub>2×</sub>):7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 6μM I 7.5μL(95◦C for 5 minutes first), total vol 30μL<br>


Set to 150 V, run for 30 minutes
==Preparation of gold-DNA nanoconjugates==


1、Prepare 10 mM TCEP.<br>
2、 Pipette 8μL of 500 mM DNA into a microcentrifuge tube<br>
3、Add 1μL of 500 mM acetate buffer (pH 5.2) and 1μL of 10 mM TCEP to the tube to activate the thiol-modified DNA. Incubate the sample at room temperature for 1 h.<br>
4、Transfer 56μL of the gold nanoparticles and 1334ul H20 to the tube, and then add the TCEP-treated thiol DNA with gentle shaking by hand.<br>
5、Cap the tube and store it in a drawer at room temperature for at least 16 h. Magnetic stirring may also be applied to facilitate the reaction.<br>
6、 Add 14μL of 500 mM Tris acetate (pH 8.2) buffer dropwise to the tube with gentle hand shaking. <br>
7、Add 14μL of 1 M NaCl dropwise to the tube with gentle hand shaking. Store the tube in a drawer for at least another day before use.<br>
8、Centrifuge the two tubes at 1500g at room temperature (23–25 °C) on a centrifuge for 60 min.<br>
9、Remove the tube from the centrifuge. The supernatant should be clear, and the nanoparticles should be at the bottom of the tubes. If a red color can still be observed in the supernatant, centrifuge for another 5 min.<br>
10、Gently pipette off as much supernatant as possible to remove free DNA. Again, disperse the nanoparticles in 30μL H<sub>2</sub>O.<br>
11、 The resulting Au-DNA conjugates were loaded on 2% agarose gels and run for one hour at 100 V. <br>




Latest revision as of 20:45, 25 October 2014

<html> <head> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <title>无标题文档</title> <style type="text/css">

body { 

    display:block;
    background-color: #ffffff;

    }

.ys01 { 

     text-align: left;
     }

/* --the header css -- */

  1. apDiv1 {

position:relative; 

       float:left;

left:0px; top:0px; 

       margin-right:0px;

width:1000px; height:350px; z-index:1; 

       box-shadow: 0px 5px 8px rgba(0,0,0,0.4);

overflow: visible; text-align: left; background-image: url(http://openwetware.org/images/3/37/Header_tianjin.png); 

       }

/* -- navigator css -- */

  1. apDiv2 {

position:relative; float:left; 

       left:0px;

width:1000px; height:40px; z-index:2; font-family: Georgia, "Times New Roman", Times, serif; text-align: left; background-image: url(http://openwetware.org/images/thumb/7/76/Tianjin_index02.jpg/800px-Tianjin_index02.jpg); 

       }
  1. apDiv2 table {

font-size: 18px; 

             text-align: center;
             }


.ys01 #apDiv2 table { text-align: center; 

                   }

.ys01 #apDiv2 table tr td a { color: #FFF; 

                           }

/* -- navigator css -- */

  1. navDiv li { margin: 0; padding: 0;}
  2. navDiv ul {margin: 0; padding: 0; list-style: none; z-index:99;}
  3. navDiv a {text-decoration: none;margin: 0; padding: 0;}
  4. navDiv

{ 

       /* -- background body of navigator css -- */

position: relative; 

       float: left;

height: 40px; width:1000px; left: 0px; background-color: #ffffff; background-repeat: repeat-x; box-shadow: 0px 5px 8px rgba(0,0,0,0.4); margin-bottom:40px; 

       z-index:60;
       font-family:  Georgia, "Times New Roman", Times, serif;
       text-align: left;

}

  1. navDiv > ul > li {
   float: left;
   margin-left: 30px;
   position: relative;

}

/* -- when mouse not on the navigator css -- */

  1. navDiv > ul > li > a {
   color: #333333;
   font-family: Verdana, 'Lucida Grande';
   font-size: 15px;
   line-height: 40px;
   padding: 11px 20px;

-webkit-transition: color .15s; 

  -moz-transition: color .15s;
    -o-transition: color .15s;
       transition: color .15s;

}

/* -- when mouse on the navigator css -- */

  1. navDiv > ul > li > a:hover {color: #ffffff; background-color: #000000;}

/* -- top and bottom of the sub meun css -- */

  1. navDiv > ul > li > ul

{ 

   opacity: 0;
   visibility: hidden;
   padding: 16px 0 20px 0;
   background-color: rgb(250,250,250);
   text-align: left;
   position: absolute;
   top: 30px;
   left: 50%;
   margin-left: -90px;
   width: 180px;

-webkit-transition: all 0.3s 0.1s; 

  -moz-transition: all 0.3s 0.1s;
    -o-transition: all 0.3s 0.1s;
       transition: all 0.3s 0.1s;

-webkit-border-radius: 5px; 

  -moz-border-radius: 5px;
       border-radius: 5px;

-webkit-box-shadow: 0px 1px 3px rgba(0,0,0,0.4); 

  -moz-box-shadow: 0px 1px 3px rgba(0,0,0,0.4);
       box-shadow: 0px 1px 3px rgba(0,0,0,0.4);

}

  1. navDiv > ul > li:hover > ul

{ 

   opacity: 1;
   top: 40px;
   visibility: visible;

}

  1. navDiv > ul > li > ul:before

{ 

   content: '';
   display: block;
   border-color: transparent transparent rgb(250,250,250) transparent;
   border-style: solid;
   border-width: 10px;
   position: absolute;
   top: -20px;
   left: 50%;
   margin-left: -10px;

}

  1. navDiv > ul ul > li { position: relative;}

/* -- when mouse not on the submeun css -- */

  1. navDiv ul ul a

{ 

   color: rgb(50,50,50);
   font-family: Verdana, 'Lucida Grande';
   font-size: 13px;
   background-color: rgb(250,250,250);
   padding: 5px 8px 7px 16px;
   display: block;

-webkit-transition: background-color .1s; 

  -moz-transition: background-color .1s;
    -o-transition: background-color .1s;
       transition: background-color .1s;

}

  1. navDiv ul ul ul

{ 

   visibility: hidden;
   opacity: 0;
   position: absolute;
   top: -16px;
   left: 206px;
   padding: 16px 0 20px 0;
   background-color: rgb(250,250,250);
   text-align: left;
   width: 160px;

-webkit-transition: all .3s; 

  -moz-transition: all .3s;
    -o-transition: all .3s;
       transition: all .3s;

-webkit-border-radius: 5px; 

  -moz-border-radius: 5px;
       border-radius: 5px;

-webkit-box-shadow: 0px 1px 3px rgba(0,0,0,.4); 

  -moz-box-shadow: 0px 1px 3px rgba(0,0,0,.4);
       box-shadow: 0px 1px 3px rgba(0,0,0,.4);

}

  1. navDiv ul ul > li:hover > ul { opacity: 1; left: 196px; visibility: visible;}

/* -- when mouse on the navigator css -- */

  1. navDiv ul ul a:hover

{ 

   background-color: #777777;
   color: rgb(240,240,240);

}


/* Back to top css */

.back-to { 

   position: fixed;
   bottom: 35px;
   *bottom: 50px;
   _bottom: 35px;
   right: 0px;
   z-index: 999;
   width: 50px;
   zoom: 1;
   }
  • html .back-to {
   /* 不能用 _position 这种写法，因为它在IE7+也会执行expression。。。 */
   position: expression(function(ele){ele.runtimeStyle.position='absolute';Expressions.style.position.fixed(ele);}(this))
   }

.back-to { 

   float: right;
   display: block;
   width: 50px;
   height: 50px;
   background: url(http://a.xnimg.cn/imgpro/button/back-home.png) no-repeat 0 0;
   outline: 0 none;
   text-indent: -9999em;

   }

.back-to:hover { 

   background-position: -50px 0
   }

.back-to .back-top { 

   float: right;
   display: block;
   width: 50px;
   height: 50px;
   background: url(http://a.xnimg.cn/imgpro/button/back-top.png) no-repeat 0 0;
   margin-left: 10px;
   outline: 0 none;
   text-indent: -9999em;
   }

.back-to .backtotop { 

   float: left;
   display: block;
   width: 50px;
   height: 50px;
   background: #666 url(http://a.xnimg.cn/imgpro/arrow/btt.png) 8px -57px no-repeat;
   margin-bottom: 15px;
   outline: 0 none;
   text-indent: -9999em;
   -moz-border-radius: 4px;
   -khtml-border-radius: 4px;
   -webkit-border-radius: 4px;
   border-radius: 4px;
   position: relative;
  -moz-box-shadow:  0px 0px 15px #ccc;
  -webkit-box-shadow:  0px 0px 15px #ccc;
  box-shadow:  0px 0px 15px #ccc;
   }

.back-to .backtotop:hover { 

   background-color: #333;
   background-position: 8px 13px;
   }

.back-to .backtotop .back-tip { 

   position: absolute;
   visibility: hidden;
   top: -31px;
   left: -10px;
   }

.back-to .backtotop:hover .back-tip { 

   visibility: visible;
   }

.back-to .back-top:hover { 

   background-position: -50px 0;
   }

/* --video css -- */

  1. apDiv3 table {

text-align: center; 

             }

.ys01 #apDiv6 table { text-align: left; 

                    }


.ys02 { 

     font-size: 36px;
     color: #F00;
     }


  1. apDiv10 {
        position:relative;

float:left; 

        display:block;
        left:0px;

width:961px; z-index:3; 

        box-shadow: 0px 5px 8px rgba(0,0,0,0.4);

text-align: left; font-family: Georgia, "Times New Roman", Times, serif; font-size: 18px; color: #000; background-color: #ffffff; 

        padding: 20px;
        }

/*hidden section*/

.firstHeading{display:none;}

  1. sidebar-main{display:none;}
  2. p-cactions{display:none;}
  3. p-personal{display:none;}

</style> </head>


<body class="ys01";>

<!--header--> <div id="apDiv1"></div>


<!--navigator starts here--> 

<div id="navDiv">

<ul> 

  	    <li class='active '><a href="http://openwetware.org/wiki/Biomod/2014/Tianjin"><span>Home</span></a></li>
  	    <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html"><span>Idea</span></a>
  	        <ul>
  	            <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#background'><span>Background</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#motivation'><span>Motivation</span></a></li>
                   <li><a href='http://openwetware.org/wiki/Biomod/2014/wiki-2%27.html#calculation'><span>Calculation</span></a></li>
 	        </ul>   
           </li>
  	    
           <li class='has-sub'><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html"><span>Project</span></a>
  	        <ul>
  	            <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Three-arm locker"><span>Three-arm Locker</span></a></li>
                   <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#Gold nanoparticle based photosensor"><span> Gold Nanoparticle Based Photosensor</span></a></li>
                   <li><a href="http://openwetware.org/wiki/Biomod/2014/experiment.html#dna origami"><span>DNA Origami</span></a></li>
               </ul> 			
           </li>   	
  	    
           <li class='active'><a href="http://openwetware.org/wiki/Tianjin_protocol"><span>Protocol</span></a>

</li> 

           <li class='active'><a href="http://openwetware.org/wiki/Biomod/2014/future.html"><span>Future Work</span></a>

</li> 

           <li class='active '><a href="http://openwetware.org/wiki/Biomod/2014/members.html"><span>Members and Acknowledgement</span></a>
           </li>

</ul> </div>


<div id="toolBackTo" class="back-to" style="left: 1175px; "> <a stats="site_footer_back_to_top" class="backtotop" href="#top" onclick="if(Sizzle('#sidebar2 .ready-to-fix')[0]) Sizzle('#sidebar2 .ready-to-fix')[0].style.position = 'static';window.scrollTo(0,0);if(Sizzle('#sidebar2 .ready-to-fix')[0]) Sizzle('#sidebar2 .ready-to-fix')[0].style.position = '';return false;">Back to top <img stats="site_footer_back_to_top" src="http://openwetware.org/images/a/a7/TJU2012-Back-tip.png" class="back-tip"> </a> </div>


<div id="apDiv10"> </html>

Buffer Preparation

(1)5×TBE: Add 54g Tris Base, 3.7224g EDTA Na2﹒2H2O and 27.5g borieacid in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL. Make the pH into 8.25～8.30.
(2)1×TBE: Add 200mL of 5×TBE in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.
(3)50×TAE: Add 242g Tris Base, 23g EDTA and 57.1mL A cetic acid glacial in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.
(4)1×TAE: Add 20mL of 50×TAE in a 1000mL volumetric flask. Add D.D. water until the final volume is 1000 mL.

Gel Pouring

PAGE Gel(10%)

Content Volume
Acrylamide Gel 30% 15ml
5×TBE 6ml
ddH2O 9ml
TEMED 20µl
ASP 150µl
Total 15ml

Prepare gel according the volumes in the table above
Pour gel into casting tray, and insert comb (make sure there are no bubbles)
Put the whole setup in 37℃ incubator for 30 min
Fill in TBE-Buffer
Prepare the sample by adding 6×loading buffer containing Golden View

Agarose Gel

1.Place 1.0(or 2.0) g of agarose in a clean 600 mL beaker on a scale.
2.Prepare the 1×TAE buffer as described.
3.Add 100 mL of 1×TAE buffer.
4.Microwave for 2 minutes.
5.If the solution is not clear, swirl and microwave for another 15 seconds.
6.Cool the beaker in an ice water bath. Keep swirling the beaker to prevent polymerization. Add the thermometer and keep checking the temperature.
7.When the solution reaches 60°C, remove it from the ice water bath.
8.Add 5µL of EB.
9.Pour the contents of the beaker into the gel cassette and insert the comb.
10.Use a glass Pasteur pipet to push any bubbles to the corners of the gel.
11.Leave the cassette covered and let the assembly sit for thirty minutes to complete ensure complete polymerization.

Origimi

Design

We want to design a two-dimensional DNA origami plate which can be folded by our locker into a roll. The dimensions of the origami plate are properly selected so the drug stays encapsulated inside it. The final dimensions are the following:
Length: 50 nm
Width: 35 nm
Thickness: 2 nm
To enable the origami to fold into a roll, the three arm locker should be combined with it. The staple strands in two corners of the origami plate are extended with the sequences of strand A, and the staple strands in the other two corners are extended with the sequences of strand C.
We used the cadnano 2 program for our design.
The scaffold used in this design is M13mp18 single strand DNA with 7249 nt, from New England Biolabs LTD. The desired structure and dimensions are achieved with the help of two types of staples:
core: 58 staples give stability to the whole structure.
edge: 4 staples give stability to the edges and with extending sequences of A or C.

Folding and Characterization

The mixture of scaffold and different staples were subjected to a thermal annealing process composed of 3 steps that allowed the folding. Different initial temperatures varying from 50.0 to 65.0 in step 2 were tested. Electrophoresis showed that low initial temperatures in step 2 have relatively high origami yield.

Different Thermal Annealing Processes of DNA Origami

Formation of the Hairpins

A, B and C did a snap-cooling procedure to form hairpin structure： heating at 90 ◦C for 5 minutes and cooling on ice for 1 minute. The hairpins were allowed to equilibrate at room temperature for 30 minutes before use. System including: 60µL ddH2O, 30µL 10µM DNA(A,B,C),10µL 10×Reaction Buffer(RB) total volume is 100µL/tube. (3µM reaction) For 6µM I: 30µL ddH2O, 60µL 10µM DNA,10µL 10×Reaction buffer

Assembly of the Three-arm Locker

Dilution oligonucleotides

Add water to 100µM. Then 10µL 100µM DNA+90µL water are mixed to get 10µM DNA.

Make A, B and C form hairpins as described.

Make A, B and C form hairpins as described.

Assembly(conc:750nM)

(1)single:7.5μL hairpin A+22.5μL 1xRB, total vol 30μL
(2)assemble: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 6μM I 7.5μL, total vol 30μL
(3)leakage: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 7.5μL 1xRB, total vol 30μL
(4)annealed: 7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 7.5μL 1xRB (95◦C for 5 minutes first) , total vol 30μL
(5)(ABCI2×):7.5μL hairpin A+7.5μL hairpin B+7.5μL hairpin C+ 6μM I 7.5μL(95◦C for 5 minutes first), total vol 30μL

Preparation of gold-DNA nanoconjugates

1、Prepare 10 mM TCEP.
2、 Pipette 8μL of 500 mM DNA into a microcentrifuge tube
3、Add 1μL of 500 mM acetate buffer (pH 5.2) and 1μL of 10 mM TCEP to the tube to activate the thiol-modified DNA. Incubate the sample at room temperature for 1 h.
4、Transfer 56μL of the gold nanoparticles and 1334ul H20 to the tube, and then add the TCEP-treated thiol DNA with gentle shaking by hand.
5、Cap the tube and store it in a drawer at room temperature for at least 16 h. Magnetic stirring may also be applied to facilitate the reaction.
6、 Add 14μL of 500 mM Tris acetate (pH 8.2) buffer dropwise to the tube with gentle hand shaking.
7、Add 14μL of 1 M NaCl dropwise to the tube with gentle hand shaking. Store the tube in a drawer for at least another day before use.
8、Centrifuge the two tubes at 1500g at room temperature (23–25 °C) on a centrifuge for 60 min.
9、Remove the tube from the centrifuge. The supernatant should be clear, and the nanoparticles should be at the bottom of the tubes. If a red color can still be observed in the supernatant, centrifuge for another 5 min.
10、Gently pipette off as much supernatant as possible to remove free DNA. Again, disperse the nanoparticles in 30μL H2O.
11、 The resulting Au-DNA conjugates were loaded on 2% agarose gels and run for one hour at 100 V.


<html>

</body> <html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Tianjin_protocol&oldid=837080"