Biomod/2014/ASU/Protocols: Difference between revisions
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</ul></li> | </ul></li> | ||
<li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Results"><i class="menu-icon icon-studio"></i><span>Results</span></a></li> | <li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Results"><i class="menu-icon icon-studio"></i><span>Results</span></a></li> | ||
<li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Discussion"><i class="menu-icon icon-studio"></i><span>Discussion</span></a></li> | |||
<li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Team"><i class="menu-icon icon-studio"></i><span>Team</span></a></li> | <li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Team"><i class="menu-icon icon-studio"></i><span>Team</span></a></li> | ||
<li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Acknowledgement"><i class="menu-icon icon-studio"></i><span>Acknowledgements</span></a></li> | <li><a href="http://openwetware.org/wiki/Biomod/2014/ASU/Acknowledgement"><i class="menu-icon icon-studio"></i><span>Acknowledgements</span></a></li> | ||
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</nav> | </nav> | ||
<div id="container" class="clearfix"> | |||
<div id = "b2x2"> | <div id = "b2x2"> | ||
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</div> | </div> | ||
</div> | </div> | ||
<div class="box b1x2" data-symbol="description" data-category="student"> | <div class="box b1x2" data-symbol="description" data-category="student"> | ||
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Tile Assembly</span></p> | <p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Tile Assembly</span></p> | ||
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<p style = "color: #000000; font-size: 16px;">2. Anneal tiles by slowly cooling from 90 °C to 25 °C over the course of 10 hours according to the following protocol:</p><br> | <p style = "color: #000000; font-size: 16px;">2. Anneal tiles by slowly cooling from 90 °C to 25 °C over the course of 10 hours according to the following protocol:</p><br> | ||
<img src = "http://openwetware.org/images/2/2f/Screen_Shot_2014-10-23_at_3.23.40_AM.png"><br> | <img src = "http://openwetware.org/images/2/2f/Screen_Shot_2014-10-23_at_3.23.40_AM.png"><br> | ||
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">4% Agarose gel electrophoresis</span></p> | |||
<hr> | |||
<p style = "color: #000000; font-size: 16px;">1. Prepare the agarose gel chamber by inserting gel walls and comb.</p> | |||
<p style = "color: #000000; font-size: 16px;">2. Add 0.2 g of agarose powder to 50 mL 0.5X TBE in an Erlenmeyer flask. Gently mix by swirling.</p> | |||
<p style = "color: #000000; font-size: 16px;">3. Microwave the agarose solution for 45 seconds on HIGH.</p> | |||
<p style = "color: #000000; font-size: 16px;">4. Add 5 μL SYBR Safe stain to the heated agarose solution.</p> | |||
<p style = "color: #000000; font-size: 16px;">5. Pour the heated gel solution into the chamber. Allow half an hour for the gel to polymerize and harden.</p> | |||
<p style = "color: #000000; font-size: 16px;">6. After the gel has polymerized, remove the temporary gel walls and fill the chamber with 0.5X TBE.</p> | |||
<p style = "color: #000000; font-size: 16px;">7. Prepare loading samples by adding one-tenth their volume of 10X native loading dye to each.</p> | |||
<p style = "color: #000000; font-size: 16px;">8. Pipet 8 μL of each sample into the sample wells.</p> | |||
<p style = "color: #000000; font-size: 16px;">9. Run at 100V for an hour and a half or until the loading dye has migrated approximately halfway through the gel.</p> | |||
<p style = "color: #000000; font-size: 16px;">10. Image the gel via UV transillumination.</p> | |||
<br> | |||
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">EMSA via Native PAGE</span></p> | <p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">EMSA via Native PAGE</span></p> | ||
<hr> | <hr> | ||
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<p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. While samples are incubating, prepare an 8% Native PAGE gel:</p> | <p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. While samples are incubating, prepare an 8% Native PAGE gel:</p> | ||
<p style = "color: #000000; font-size: 16px;"> a. Set up the gel assembly by aligning glass plates and spacers, tightening the screws of the clamps, screwing the assembly down</p> | <p style = "color: #000000; font-size: 16px;"> a. Set up the gel assembly by aligning glass plates and spacers, tightening the screws of the clamps, screwing the assembly down</p> | ||
<p style = "color: #000000; font-size: 16px;"> into a rubber pad, and inserting a comb.</p> | |||
<p style = "color: #000000; font-size: 16px;"> b. Mix 28 mL H2O, 4 mL 10x TAE/Mg2+ + 1M KCl buffer, and 8 mL 40% acrylamide in an Erlenmeyer flask. Swirl to mix.</p> | <p style = "color: #000000; font-size: 16px;"> b. Mix 28 mL H2O, 4 mL 10x TAE/Mg2+ + 1M KCl buffer, and 8 mL 40% acrylamide in an Erlenmeyer flask. Swirl to mix.</p> | ||
<p style = "color: #000000; font-size: 16px;"> c. Add 16.8 μL TEMED and 300 μL APS. Swirl to mix. </p> | <p style = "color: #000000; font-size: 16px;"> c. Add 16.8 μL TEMED and 300 μL APS. Swirl to mix. </p> | ||
<p style = "color: #000000; font-size: 16px;"> d. Use a serological pipette to fill the assembly with gel solution. Allow 40 minutes for polymerization.</p> | <p style = "color: #000000; font-size: 16px;"> d. Use a serological pipette to fill the assembly with gel solution. Allow 40 minutes for polymerization.</p> | ||
<p style = "color: #000000; font-size: 16px;"> e. Remove the comb and attach an upper buffer chamber to the gel assembly and to a second, solid glass plate filler.</p> | <p style = "color: #000000; font-size: 16px;"> e. Remove the comb and attach an upper buffer chamber to the gel assembly and to a second, solid glass plate filler.</p> | ||
<p style = "color: #000000; font-size: 16px;"> f. Fill buffer chamber with 500 mL 1x TAE/Mg2+ + 100 mM KCl. Rinse out wells of gel with ambient buffer using a needled syringe.</p> | <p style = "color: #000000; font-size: 16px;"> f. Fill buffer chamber with 500 mL 1x TAE/Mg2+ + 100 mM KCl. Rinse out wells of gel with ambient buffer using a needled</p> | ||
<p style = "color: #000000; font-size: 16px;"> syringe.</p> | |||
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Load and run the gel:</p> | <p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Load and run the gel:</p> | ||
<p style = "color: #000000; font-size: 16px;"> a. Load 10 μL sample into different wells of the gel.</p> | <p style = "color: #000000; font-size: 16px;"> a. Load 10 μL sample into different wells of the gel.</p> | ||
<p style = "color: #000000; font-size: 16px;"> b. Put the completed gel assembly into a lower buffer chamber with 1x TAE/Mg2+. Connect it to a voltage generator, and run the gel at 200 V for 8 hours.</p> | <p style = "color: #000000; font-size: 16px;"> b. Put the completed gel assembly into a lower buffer chamber with 1x TAE/Mg2+. Connect it to a voltage generator, and run the</p> | ||
<p style = "color: #000000; font-size: 16px;"> gel at 200 V for 8 hours.</p> | |||
<p style = "color: #000000; font-size: 16px;"> c. After the gel has run for four hours, refresh the upper buffer chamber with 500 mL of fresh 1x TAE/Mg2+ + 100 mM KCl buffer.</p> | <p style = "color: #000000; font-size: 16px;"> c. After the gel has run for four hours, refresh the upper buffer chamber with 500 mL of fresh 1x TAE/Mg2+ + 100 mM KCl buffer.</p> | ||
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">4. Stain the gel:</p> | <p style = "color: #000000; font-size: 16px;text-decoration:underline;">4. Stain the gel:</p> | ||
<p style = "color: #000000; font-size: 16px;"> a. Mix 10 uL 10000x SYBR Green with 100 mL water in a clean staining box.</p> | <p style = "color: #000000; font-size: 16px;"> a. Mix 10 uL 10000x SYBR Green with 100 mL water in a clean staining box.</p> | ||
<p style = "color: #000000; font-size: 16px;"> b. Disassemble the gel apparatus, and place the gel in the stain. Briefly and gently rock the staining box to ensure the gel is being evenly stained. Allow the gel to stain for 5 minutes.</p> | <p style = "color: #000000; font-size: 16px;"> b. Disassemble the gel apparatus, and place the gel in the stain. Briefly and gently rock the staining box to ensure the gel is being </p> | ||
<p style = "color: #000000; font-size: 16px;"> evenly stained. Allow the gel to stain for 5 minutes.</p> | |||
<p style = "color: #000000; font-size: 16px;"> c. After staining, briefly place the gel in water to destain.</p> | <p style = "color: #000000; font-size: 16px;"> c. After staining, briefly place the gel in water to destain.</p> | ||
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">5. Image the gel:</p> | <p style = "color: #000000; font-size: 16px;text-decoration:underline;">5. Image the gel:</p> | ||
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<p style = "color: #000000; font-size: 16px;"> 5. 72 °C for 5 minutes</p> | <p style = "color: #000000; font-size: 16px;"> 5. 72 °C for 5 minutes</p> | ||
<p style = "color: #000000; font-size: 16px;"> 6. 4 °C forever</p><br> | <p style = "color: #000000; font-size: 16px;"> 6. 4 °C forever</p><br> | ||
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Alpha-Thrombin-bound NHS ester bead affinity column:</span></p> | <p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Alpha-Thrombin-bound NHS ester bead affinity column:</span></p> | ||
<hr> | <hr> | ||
<p style = "color: #000000; font-size: 16px | <p style = "color: #000000; font-size: 16px;"><u>1. Prepare columns by functionalizing Pierce® NHS-Activated Agarose Dry Resin with alpha-thrombin:</u></p> | ||
<p style = "color: #000000; font-size: 16px;"> a. In one pot, measure out 5 mg NHS | <p style = "color: #000000; font-size: 16px;"> a. In one pot, measure out 5 mg Pierce® NHS-Activated Agarose Dry Resin per each column to </p> | ||
<p style = "color: #000000; font-size: 16px;"> b. Dilute | <p style = "color: #000000; font-size: 16px;"> be prepared.</p> | ||
<p style = "color: #000000; font-size: 16px;"> b. Dilute alpha-thrombin stock into a 1 mg/mL 0.1 M HEPES + 0.15 M NaCl buffered solution with 30 μL volume per each column to be prepared.</p> | |||
<p style = "color: #000000; font-size: 16px;"> c. Combine entire diluted alpha-thrombin stock with weighed-out Pierce® NHS-Activated Agarose Dry Resin.</p> | |||
<p style = "color: #000000; font-size: 16px;"> d. Mix end-over-end for one hour in a filter column.</p> | |||
<p style = "color: #000000; font-size: 16px;"> e. Centrifuge at 1000 rfc for 1 minute. Save flowthrough.</p> | |||
<p style = "color: #000000; font-size: 16px;"> f. Add 30 μL volume per column to be prepared of 0.1 M HEPES, 0.15 M NaCl buffer. Recentrifuge at 1000 rfc for 1 minute and save </p> | |||
<p style = "color: #000000; font-size: 16px;"> flowthrough.</p> | |||
<p style = "color: #000000; font-size: 16px;"> g. Add 30 μL per column to be prepared of Tris buffer at pH 8 to quench column and mix end-over-end for 15-20 minutes at room</p> | |||
<p style = "color: #000000; font-size: 16px;"> temperature.</p> | |||
<p style = "color: #000000; font-size: 16px;"> h. Centrifuge at 1000 rfc for 1 minute and save flowthrough.</p> | |||
<p style = "color: #000000; font-size: 16px;"> i. Add 30 μL volume per column to be prepared of 0.1 M HEPES + 0.15 M NaCl + 0.05% NaN3 buffer to resuspend resin after </p> | |||
<p style = "color: #000000; font-size: 16px;"> quenching.</p> | |||
<p style = "color: #000000; font-size: 16px;"> j. Split mix into columns with 30 μL suspended beads each and store at 4 °C.</p> | |||
<p style = "color: #000000; font-size: 16px;"><u>2. Run column assay:</u></p> | |||
<p style = "color: #000000; font-size: 16px;"> a. Prepare a 1 μM loading solution of the desired sample in 20 μL of a 1x TAE + 0.1M KCl buffered solution.</p> | |||
<p style = "color: #000000; font-size: 16px;"> b. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting. Centrifuge at 1000 rfc for 1 minute.</p> | |||
<p style = "color: #000000; font-size: 16px;"> c. Add 20 μL loading solution to column. Mix end-over-end for 1 hour.</p> | |||
<p style = "color: #000000; font-size: 16px;"> d. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p> | |||
<p style = "color: #000000; font-size: 16px;"> e. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting.</p> | |||
<p style = "color: #000000; font-size: 16px;"> f. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p> | |||
<p style = "color: #000000; font-size: 16px;"> g. Repeat steps e-f 3 more times. </p> | |||
<p style = "color: #000000; font-size: 16px;"> h. Add 60 μL 1x TAE + 0.1M KCl to column, mix by gently pipetting, and pipet the contents of the column into an Eppendorf tube for </p> | |||
<p style = "color: #000000; font-size: 16px;"> storage.</p> | |||
<p style = "color: #000000; font-size: 16px;">Atomic Force Microscopy was performed by Shuoxing Jiang, a member of the Hao Yan Lab, using the Dimension FastScan AFM (Bruker). 2μl of sample was deposited onto freshly cleaved mica and left to absorb for 2 minutes. 100μl of 1x TAE Mg was pipetted on top of the DNA sample and the sample was imaged in ScanAsyst in Fluid mode using a ScanAssyst Fluid+ tip.</p> | |||
Latest revision as of 23:58, 25 October 2014
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border: 1px solid rgba(0, 0, 0, 0.3);
height: 150px;
position: relative;
z-index: 3;
}
.interlude *{
float:left;
}
.interlude h2{
color: #FFFFFF;
display: block;
font-weight: 300;
line-height: 150px;
margin-left: 24%;
margin-right: -14%;
width: 50%;
}
.interlude img{
float: left;
line-height: 150px;
margin-left: 10%;
margin-top: 25px;
vertical-align: middle;
}
.clear{
clear: both;
}
.project_box{
background: none repeat scroll 0 0 #FFFFFF;
color: #000000;
display: block;
float: left;
font-size: 18px;
font-weight: 300;
line-height: 1.6;
margin-left: auto;
margin-right: auto;
overflow: hidden;
padding: 10px;
width: 50%;
}
.figure_box{
display: block;
float: left;
margin-left: 20px;
overflow: hidden;
width: 230px;
}
/*se*/
.project_box h2{
color: #1A1A1A;
display: block;
font-weight: 300;
margin-left: 0%;
margin-right: -10%;
width: 90%;
}
.project_box p{
text-align: justify;
margin-bottom: 18px;
}
.project_box li {
margin-left:50px
}
- pb_mot.project_box{
height: 350px;
}
- pb_dna_scaff.project_box{
height: 400px;
}
- pb_dna_req.project_box{
height: 250px;
}
- pb_poly_intro.project_box{
/*right: -20%;*/
height: 200px;
}
- pb_poly_pmoxa.project_box{
/*right: -20%;*/
height: 600px;
width: 1000px;
overflow:scroll;
}
- pb_ir.project_box{
/*right: -20%;*/
height: 1300px;
}
- pb_poly_cp.project_box{
/*right: -20%;*/
height: 600px;
}
/*team page*/
.bio_box {
background: none repeat scroll 0 0 #E74C3C;
float: left;
font-size: 15px;
height: 440px;
padding: 15px;
text-align: justify;
width: 210px;
}
.bio_box > .name{
font-size: 24px;
font-weight: 300;
margin-bottom: 25px;
text-align: center;
width: 100%;
}
.bio_box > p{
text-align: justify;
font-weight: 300;
font-size: 16px;
}
.box.big img{
opacity:1;
}
.flag > *{
float:left;
}
.flag > p{
font-size: 18px;
position: relative;
text-align: center;
top: -6px;
width: 75%;
margin-bottom: 10px;
}
- team .big{
opacity:1;
}
.head{
width:220px;
float:left;
}
/*sponsor page*/
- sponsors .box {
background: none repeat scroll 0 0 white;
}
- sponsors figcaption {
height: 65px;
width: 100%;
font-size: 15px;
font-weight: 300;
top: auto;
bottom: 0;
opacity: 0;
transform: translateY(100%);
transition: transform 0.4s, opacity 0.1s 0.3s;
-webkit-transform: translateY(100%);
-webkit-transition: -webkit-transform 0.4s, opacity 0.1s 0.3s;
}
- sponsors .descr{
background: none repeat scroll 0 0 rgba(0, 0, 0, 0.4);
font-size: 12px;
font-weight: 300;
height: 60px;
margin: 0;
padding-left: 10px;
padding-right: 10px;
padding-top: 10px;
text-align: justify;
top: -155px;
line-height: 1.3;
}
- sponsors .descr p{
width:90%;
margin-left:auto;
margin-right:auto;
}
- sponsors figure.clickable:hover figcaption{
opacity: 1;
transform: translateY(0px);
transition: transform 0.4s, opacity 0.1s;
-webkit-transform: translateY(0px);
-webkit-transition: -webkit-transform 0.4s, opacity 0.1s;
}
- sponsors figure:hover .descr{
opacity: 1;
transform: translateY(155px);
transition: transform 0.4s, opacity 0.1s;
-webkit-transform: translateY(155px);
-webkit-transition: -webkit-transform 0.4s, opacity 0.1s;
}
/*gallery*/
- gallery .box img{
min-height: 220px;
min-width: 220px;
}
/*ptocols*/
.protocol_box{
background: none repeat scroll 0 0 #FFFFFF;
color: #000000;
display: block;
float: left;
font-size: 18px;
font-weight: 300;
line-height: 1.6;
margin-left: 40px;
margin-right: auto;
overflow: hidden;
padding: 10px;
width: 66%;
}
.protocol_box li {
margin-left:50px
}
.protocol_box p{
text-align: justify;
margin-top: 18px;
}
.protocol_box h1 {
font-size: 30px;
}
.protocol_box h2 {
font-size: 24px;
}
.protocol_box h3 {
font-size: 22px;
}
/*Outreach*/
.outreach_box{
background: none repeat scroll 0 0 #FFFFFF;
color: #000000;
display: block;
float: left;
font-size: 18px;
font-weight: 300;
line-height: 1.6;
margin-left: 10px;
margin-right: auto;
overflow: hidden;
padding: 10px;
width: 70%;
}
.outreach_box li {
margin-left:50px
}
/*Acknowlegement*/
.ack_box{
background: none repeat scroll 0 0 #FFFFFF;
color: #000000;
text-align: center;
display: block;
float: left;
font-size: 18px;
font-weight: 300;
line-height: 1.6;
margin-left: 10px;
margin-right: auto;
overflow: hidden;
padding: 10px;
width: 80%;
}
.ack_box p {
text-align: center;
}
.next, .prev{
z-index: 99;
background-image: url("http://openwetware.org/images/5/55/Fancybox_sprite.png");
width: 36px;
height: 36px;
top: 200px;
}
figure.box > .next {
left: 425px;
background-position: 0 -72px;
}
figure.box > .prev {
background-position: 0 -36px;
}
/**** Isotope styles ****/
/* required for containers to inherit vertical size from window */
html,
body {
height: 100%;
}
- container {
padding: 0px;
bottom-margin: 10px;
}
.box {
width: 220px;
height: 220px;
margin: 10px;
float: left;
overflow: hidden;
position: fixed;
background: #fff;
color: #000000;
display: table-cell;
text-align: center;
vertical-align: middle;
overflow:hidden;
}
div#b2x2{ position:fixed;}
figure.box > *{
left: 0;
position: absolute;
right: 0;
}
.box figure{
overflow: hidden;
}
.box figcaption {
background: none repeat scroll 0 0 rgba(0, 0, 0, 0.4);
bottom: 0;
font-size: 20px;
font-weight: 300;
padding-left: 5px;
text-align: center;
width: 100%;
z-index: 4;
}
.clickable .box:hover {
cursor: pointer;
}
/* The Magnificent Clearfix: nicolasgallagher.com/micro-clearfix-hack/ */
.clearfix:before, .clearfix:after { content: ""; display: table; }
.clearfix:after { clear: both; }
.clearfix { zoom: 1; }
/* Start: Recommended Isotope styles */
/**** Isotope Filtering ****/
.isotope-item {
z-index: 2;
}
.isotope-hidden.isotope-item {
pointer-events: none;
z-index: 1;
}
/**** Isotope CSS3 transitions ****/
.isotope,
.isotope .isotope-item {
-webkit-transition-duration: 0.8s;
-moz-transition-duration: 0.8s;
-ms-transition-duration: 0.8s;
-o-transition-duration: 0.8s;
transition-duration: 0.8s;
}
.isotope {
-webkit-transition-property: height, width;
-moz-transition-property: height, width;
-ms-transition-property: height, width;
-o-transition-property: height, width;
transition-property: height, width;
}
.isotope .isotope-item {
-webkit-transition-property: -webkit-transform, opacity;
-moz-transition-property: -moz-transform, opacity;
-ms-transition-property: -ms-transform, opacity;
-o-transition-property: -o-transform, opacity;
transition-property: transform, opacity;
}
.rs-wrap:after,
.rs-slider:after,
.rs-thumb-wrap:after,
.rs-arrows:after,
.rs-caption:after {
content: ".";
display: block;
height: 0;
clear: both;
line-height: 0;
visibility: hidden;
}
/* ===[ Slider ]=== */
.rs-wrap {
position: relative;
max-width: 100%;
}
.rs-slide-bg { *zoom: 1 }
.rs-slider > li > a { display: block }
.rs-slider > li {
list-style: none;
filter: alpha(opacity=0);
opacity: 0;
width: 100%;
height: 100%;
margin: 0 -100% 0 0;
padding: 0;
float: left;
position: relative;
}
.rs-slider > li > a {
padding: 0;
background: none;
-webkit-border-radius: 0;
-moz-border-radius: 0;
border-radius: 0;
}
.rs-slider > li img {
display: block;
max-width: 100%;
max-height: 100%;
-ms-interpolation-mode: bicubic;
}
/* ===[ Thumbnails ]=== */
.rs-thumb-wrap { *zoom: 1 }
.rs-thumb-wrap > a {
display: block;
float: left;
position: relative;
-moz-box-sizing: border-box;
-webkit-box-sizing: border-box;
box-sizing: border-box;
-webkit-backface-visibility: hidden; /* Hardware accelerate to prevent jumps on transition */
}
.rs-thumb-wrap > a > img {
max-width: 100%;
max-height: 100%;
display: block;
-ms-interpolation-mode: bicubic;
}
.rs-thumb-wrap > a:first-child { margin-left: 0!important }
/* ===[ Arrows ]=== */
.rs-arrows .rs-next,
.rs-arrows .rs-prev { z-index: 1; background-image: url("fancybox_sprite.png");}
.rs-arrows .rs-next,
.rs-arrows .rs-prev { z-index: 1; background-image: url("fancybox_sprite.png");}
.rs-arrows:hover .rs-next,
.rs-arrows:hover .rs-prev { z-index: 2; }
/* ===[ Captions ]=== */
.rs-caption {
position: absolute;
max-height: 100%;
overflow: auto;
-moz-box-sizing: border-box;
-webkit-box-sizing: border-box;
box-sizing: border-box;
bottom: 0;
left: 0;
}
.rs-caption.rs-top-left {
top: 0;
bottom: auto;
}
.rs-caption.rs-top-right {
top: 0;
right: 0;
left: auto;
bottom: auto;
}
.rs-caption.rs-bottom-left {
bottom: 0;
left: 0;
}
.rs-caption.rs-bottom-right {
right: 0;
left: auto;
border-bottom: none;
border-right: none;
}
.rs-caption.rs-top {
top: 0;
bottom: auto;
width: 100%!important;
}
.rs-caption.rs-bottom { width: 100%!important }
.rs-caption.rs-left {
top: 0;
height: 100%;
}
.rs-caption.rs-right {
top: 0;
left: auto;
right: 0;
height: 100%;
}
/* ===[ Grid ]=== */
.rs-grid {
position: absolute;
overflow: hidden;
width: 100%;
height: 100%;
display: none;
}
.rs-gridlet {
position: absolute;
opacity: 1;
}
/* Optional - remove captions at smaller screen widths
@media screen and (max-width: 480px) {
.rs-caption { opacity: 0!important; }
}
- /
.project_box > img {
margin-left: 90px;
}
- protocols, #polymers_protocols, #origami_protocols, #reaction_protocols, #nanocontainer_protocols, #imaging_protocols {
font-size: 20px;
font-weight: 300;
margin-bottom: 30px;
margin-left: 50px;
}
- protocols > h2, #polymers_protocols > h2, #origami_protocols > h2, #reaction_protocols > h2, #nanocontainer_protocols > h2, #imaging_protocols > h2 {
margin-bottom: 20px;
margin-top: 20px;
}
- protocols .interlude, #polymers_protocols .interlude, #origami_protocols .interlude, #reaction_protocols .interlude, #nanocontainer_protocols .interlude, #imaging_protocols .interlude {
margin-left: -50px !important;
}
- protocols > ul {
margin-bottom: 30px;
margin-left: 30px;
margin-top: 20px;
}
li > ul {
margin-left: 10px;
}
/*achievement*/
.achievement_box{
background: none repeat scroll 0 0 #FFFFFF;
color: #000000;
display: block;
float: left;
font-size: 18px;
font-weight: 300;
line-height: 1.6;
margin-left: 180px;
margin-right: auto;
overflow: hidden;
padding: 10px;
width: 50%;
}
- subnav-sticky-wrapper {
height: 5px !important;
}
table {
border-collapse: collapse;
margin: auto auto 40px;
width: 635px;;
}
th {
background-color: #5F5F5F;
border: 1px solid #999999;
color: #FFFFFF;
}
tr td {
border: 1px solid #999999;
text-align: center;
}
tr.odd td {
background-color: #EEEEEE;
color: #000000;
}
.ref li {
font-size: 14px;
font-weight: 300;
}
</style>
<link href="http://openwetware.org/images/2/29/Nano_icon.png" rel="shortcut icon">
<script src="https://biomod2013.googlecode.com/svn/trunk/js/jquery.isotope.min.js"></script>
<script src="https://biomod2013.googlecode.com/svn/trunk/js/jquery.refineslide.min.js"></script>
<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/fb/jquery.fancybox.pack.js"></script>
<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/fb/helpers/jquery.fancybox-buttons.js"></script>
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<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/fb/helpers/jquery.fancybox-thumbs.js"></script>
<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/jquery.easing.min.js"></script>
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<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/jquery.sticky.js"></script>
<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/jquery.scrollTo.min.js"></script>
<script type="text/javascript" src="http://biomod2013.googlecode.com/svn/trunk/js/jquery.localscroll.min.js"></script>
<script>
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ga('send', 'pageview');
</script>
<style type="text/css">
/*! fancyBox v2.1.5 fancyapps.com | fancyapps.com/fancybox/#license */
.fancybox-wrap,
.fancybox-skin,
.fancybox-outer,
.fancybox-inner,
.fancybox-image,
.fancybox-wrap iframe,
.fancybox-wrap object,
.fancybox-nav,
.fancybox-nav span,
.fancybox-tmp
{
padding: 0;
margin: 0;
border: 0;
outline: none;
vertical-align: top;
}
.fancybox-wrap {
position: absolute;
top: 0;
left: 0;
z-index: 8020;
}
.fancybox-skin {
position: relative;
background: #f9f9f9;
color: #444;
text-shadow: none;
-webkit-border-radius: 4px;
-moz-border-radius: 4px;
border-radius: 4px;
}
.fancybox-opened {
z-index: 8030;
}
.fancybox-opened .fancybox-skin {
-webkit-box-shadow: 0 10px 25px rgba(0, 0, 0, 0.5);
-moz-box-shadow: 0 10px 25px rgba(0, 0, 0, 0.5);
box-shadow: 0 10px 25px rgba(0, 0, 0, 0.5);
}
.fancybox-outer, .fancybox-inner {
position: relative;
}
.fancybox-inner {
overflow: hidden;
}
.fancybox-type-iframe .fancybox-inner {
-webkit-overflow-scrolling: touch;
}
.fancybox-error {
color: #444;
font: 14px/20px "Helvetica Neue",Helvetica,Arial,sans-serif;
margin: 0;
padding: 15px;
white-space: nowrap;
}
.fancybox-image, .fancybox-iframe {
display: block;
width: 100%;
height: 100%;
}
.fancybox-image {
max-width: 100%;
max-height: 100%;
}
- fancybox-loading, .fancybox-close, .fancybox-prev span, .fancybox-next span {
background-image: url('http://openwetware.org/images/5/55/Fancybox_sprite.png');
}
- fancybox-loading {
position: fixed;
top: 50%;
left: 50%;
margin-top: -22px;
margin-left: -22px;
background-position: 0 -108px;
opacity: 0.8;
cursor: pointer;
z-index: 8060;
}
- fancybox-loading div {
width: 44px;
height: 44px;
background: url('http://openwetware.org/images/d/d0/Fancybox_loading.gif') center center no-repeat;
}
.fancybox-close {
position: absolute;
top: -18px;
right: -18px;
width: 36px;
height: 36px;
cursor: pointer;
z-index: 8040;
}
.fancybox-nav {
position: absolute;
top: 0;
width: 40%;
height: 100%;
cursor: pointer;
text-decoration: none;
background: transparent url('http://openwetware.org/images/c/c0/Blank.gif'); /* helps IE */
-webkit-tap-highlight-color: rgba(0,0,0,0);
z-index: 8040;
}
.fancybox-prev {
left: 0;
}
.fancybox-next {
right: 0;
}
.fancybox-nav span {
position: absolute;
top: 50%;
width: 36px;
height: 34px;
margin-top: -18px;
cursor: pointer;
z-index: 8040;
visibility: hidden;
}
.fancybox-prev span {
left: 10px;
background-position: 0 -36px;
}
.fancybox-next span {
right: 10px;
background-position: 0 -72px;
}
.fancybox-nav:hover span {
visibility: visible;
}
.fancybox-tmp {
position: absolute;
top: -99999px;
left: -99999px;
visibility: hidden;
max-width: 99999px;
max-height: 99999px;
overflow: visible !important;
}
/* Overlay helper */
.fancybox-lock {
overflow: hidden !important;
width: auto;
}
.fancybox-lock body {
overflow: hidden !important;
}
.fancybox-lock-test {
overflow-y: hidden !important;
}
.fancybox-overlay {
position: absolute;
top: 0;
left: 0;
overflow: hidden;
display: none;
z-index: 8010;
background: url('http://openwetware.org/images/e/e0/Fancybox_overlay.png');
}
.fancybox-overlay-fixed {
position: fixed;
bottom: 0;
right: 0;
}
.fancybox-lock .fancybox-overlay {
overflow: auto;
overflow-y: scroll;
}
/* Title helper */
.fancybox-title {
visibility: hidden;
font: normal 13px/20px "Helvetica Neue",Helvetica,Arial,sans-serif;
position: relative;
text-shadow: none;
z-index: 8050;
}
.fancybox-opened .fancybox-title {
visibility: visible;
}
.fancybox-title-float-wrap {
position: absolute;
bottom: 0;
right: 50%;
margin-bottom: -35px;
z-index: 8050;
text-align: center;
}
.fancybox-title-float-wrap .child {
display: inline-block;
margin-right: -100%;
padding: 2px 20px;
background: transparent; /* Fallback for web browsers that doesn't support RGBa */
background: rgba(0, 0, 0, 0.8);
-webkit-border-radius: 15px;
-moz-border-radius: 15px;
border-radius: 15px;
text-shadow: 0 1px 2px #222;
color: #FFF;
font-weight: bold;
line-height: 24px;
white-space: nowrap;
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.fancybox-title-outside-wrap {
position: relative;
margin-top: 10px;
color: #fff;
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.fancybox-title-inside-wrap {
padding-top: 10px;
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.fancybox-title-over-wrap {
position: absolute;
bottom: 0;
left: 0;
color: #fff;
padding: 10px;
background: #000;
background: rgba(0, 0, 0, .8);
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/*Retina graphics!*/
@media only screen and (-webkit-min-device-pixel-ratio: 1.5),
only screen and (min--moz-device-pixel-ratio: 1.5),
only screen and (min-device-pixel-ratio: 1.5){
#fancybox-loading, .fancybox-close, .fancybox-prev span, .fancybox-next span {
background-image: url('http://openwetware.org/images/b/b8/Fancybox_sprite%402x.png');
background-size: 44px 152px; /*The size of the normal image, half the size of the hi-res image*/
}
#fancybox-loading div {
background-image: url('http://openwetware.org/images/0/01/Fancybox_loading%402x.gif');
background-size: 24px 24px; /*The size of the normal image, half the size of the hi-res image*/
}
}
- fancybox-buttons {
position: fixed;
left: 0;
width: 100%;
z-index: 8050;
}
- fancybox-buttons.top {
top: 10px;
}
- fancybox-buttons.bottom {
bottom: 10px;
}
- fancybox-buttons ul {
display: block;
width: 166px;
height: 30px;
margin: 0 auto;
padding: 0;
list-style: none;
border: 1px solid #111;
border-radius: 3px;
-webkit-box-shadow: inset 0 0 0 1px rgba(255,255,255,.05);
-moz-box-shadow: inset 0 0 0 1px rgba(255,255,255,.05);
box-shadow: inset 0 0 0 1px rgba(255,255,255,.05);
background: rgb(50,50,50);
background: -moz-linear-gradient(top, rgb(68,68,68) 0%, rgb(52,52,52) 50%, rgb(41,41,41) 50%, rgb(51,51,51) 100%);
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,rgb(68,68,68)), color-stop(50%,rgb(52,52,52)), color-stop(50%,rgb(41,41,41)), color-stop(100%,rgb(51,51,51)));
background: -webkit-linear-gradient(top, rgb(68,68,68) 0%,rgb(52,52,52) 50%,rgb(41,41,41) 50%,rgb(51,51,51) 100%);
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background: -ms-linear-gradient(top, rgb(68,68,68) 0%,rgb(52,52,52) 50%,rgb(41,41,41) 50%,rgb(51,51,51) 100%);
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margin: 0;
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display: block;
width: 30px;
height: 30px;
text-indent: -9999px;
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<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Tile Assembly</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;">1. Put all relevant tile strands into a 1x TAE/Mg2+ buffered solution at 1 μM concentration.</p>
<p style = "color: #000000; font-size: 16px;">2. Anneal tiles by slowly cooling from 90 °C to 25 °C over the course of 10 hours according to the following protocol:</p><br>
<img src = "http://openwetware.org/images/2/2f/Screen_Shot_2014-10-23_at_3.23.40_AM.png"><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">4% Agarose gel electrophoresis</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;">1. Prepare the agarose gel chamber by inserting gel walls and comb.</p>
<p style = "color: #000000; font-size: 16px;">2. Add 0.2 g of agarose powder to 50 mL 0.5X TBE in an Erlenmeyer flask. Gently mix by swirling.</p>
<p style = "color: #000000; font-size: 16px;">3. Microwave the agarose solution for 45 seconds on HIGH.</p>
<p style = "color: #000000; font-size: 16px;">4. Add 5 μL SYBR Safe stain to the heated agarose solution.</p>
<p style = "color: #000000; font-size: 16px;">5. Pour the heated gel solution into the chamber. Allow half an hour for the gel to polymerize and harden.</p>
<p style = "color: #000000; font-size: 16px;">6. After the gel has polymerized, remove the temporary gel walls and fill the chamber with 0.5X TBE.</p>
<p style = "color: #000000; font-size: 16px;">7. Prepare loading samples by adding one-tenth their volume of 10X native loading dye to each.</p>
<p style = "color: #000000; font-size: 16px;">8. Pipet 8 μL of each sample into the sample wells.</p>
<p style = "color: #000000; font-size: 16px;">9. Run at 100V for an hour and a half or until the loading dye has migrated approximately halfway through the gel.</p>
<p style = "color: #000000; font-size: 16px;">10. Image the gel via UV transillumination.</p>
<br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">EMSA via Native PAGE</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">1. Prepare loading samples</p>
<p style = "color: #000000; font-size: 16px;"> a. Prepare 10 μL samples of 20 nM DNA tile + 1 μM alpha-thrombin in a 1x TAE/Mg2+ +100 mM KCl buffered solution.</p>
<p style = "color: #000000; font-size: 16px;"> b. Prepare a negative control 10 μL sample of 20 nM DNA tile in a 1x TAE/Mg2+ + 100 mM KCl buffered solution for each</p>
<p style = "color: #000000; font-size: 16px;"> experimental sample above.</p>
<p style = "color: #000000; font-size: 16px;"> c. Allow to incubate on the benchtop at 25 °C for 1 hour. </p>
<p style = "color: #000000; font-size: 16px;"> d. Add 1.1 μL 10x native dye to each sample and mix gently with a pipette.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. While samples are incubating, prepare an 8% Native PAGE gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Set up the gel assembly by aligning glass plates and spacers, tightening the screws of the clamps, screwing the assembly down</p>
<p style = "color: #000000; font-size: 16px;"> into a rubber pad, and inserting a comb.</p>
<p style = "color: #000000; font-size: 16px;"> b. Mix 28 mL H2O, 4 mL 10x TAE/Mg2+ + 1M KCl buffer, and 8 mL 40% acrylamide in an Erlenmeyer flask. Swirl to mix.</p>
<p style = "color: #000000; font-size: 16px;"> c. Add 16.8 μL TEMED and 300 μL APS. Swirl to mix. </p>
<p style = "color: #000000; font-size: 16px;"> d. Use a serological pipette to fill the assembly with gel solution. Allow 40 minutes for polymerization.</p>
<p style = "color: #000000; font-size: 16px;"> e. Remove the comb and attach an upper buffer chamber to the gel assembly and to a second, solid glass plate filler.</p>
<p style = "color: #000000; font-size: 16px;"> f. Fill buffer chamber with 500 mL 1x TAE/Mg2+ + 100 mM KCl. Rinse out wells of gel with ambient buffer using a needled</p>
<p style = "color: #000000; font-size: 16px;"> syringe.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Load and run the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Load 10 μL sample into different wells of the gel.</p>
<p style = "color: #000000; font-size: 16px;"> b. Put the completed gel assembly into a lower buffer chamber with 1x TAE/Mg2+. Connect it to a voltage generator, and run the</p>
<p style = "color: #000000; font-size: 16px;"> gel at 200 V for 8 hours.</p>
<p style = "color: #000000; font-size: 16px;"> c. After the gel has run for four hours, refresh the upper buffer chamber with 500 mL of fresh 1x TAE/Mg2+ + 100 mM KCl buffer.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">4. Stain the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Mix 10 uL 10000x SYBR Green with 100 mL water in a clean staining box.</p>
<p style = "color: #000000; font-size: 16px;"> b. Disassemble the gel apparatus, and place the gel in the stain. Briefly and gently rock the staining box to ensure the gel is being </p>
<p style = "color: #000000; font-size: 16px;"> evenly stained. Allow the gel to stain for 5 minutes.</p>
<p style = "color: #000000; font-size: 16px;"> c. After staining, briefly place the gel in water to destain.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">5. Image the gel:</p>
<p style = "color: #000000; font-size: 16px;"> a. Place the gel on the transilluminating plate of the gel imager.</p>
<p style = "color: #000000; font-size: 16px;"> b. Image the gel using UV transillumination.</p><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">PCR</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">1. Prepare a PCR master mix solution with 2 extra equivalents. For 20 μL reactions, add, for each equivalent:</p>
<p style = "color: #000000; font-size: 16px;"> a. 4 μL 5x Phusion buffer.</p>
<p style = "color: #000000; font-size: 16px;"> b. 0.4 μL 10 mM dNTPs.</p>
<p style = "color: #000000; font-size: 16px;"> c. 0.6 μL DMSO.</p>
<p style = "color: #000000; font-size: 16px;"> d. Forward and reverse primers to 1 μM.</p>
<p style = "color: #000000; font-size: 16px;"> e. 0.2 μL Phusion High-Fidelity DNA polymerase (keep on ice)</p>
<p style = "color: #000000; font-size: 16px;"> i. Add polymerase last and keep all mixtures containing polymerase on ice thereafter.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">2. Add 2 μL of each target oligonucleotide source to 18 μL of master mix for each reaction. Keep on ice.</p>
<p style = "color: #000000; font-size: 16px;text-decoration:underline;">3. Except for a PCR-negative control, which is kept at 4 °C for the duration of thermocycling, cycle all reactions according to the following protocol:</p>
<p style = "color: #000000; font-size: 16px;"> 1. 98 °C for 1 minute</p>
<p style = "color: #000000; font-size: 16px;"> 2. 98 °C for 5 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 3. 42 °C for 10 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 4. 72 °C for 15 seconds</p>
<p style = "color: #000000; font-size: 16px;"> 4. Cycle steps 2-4 for 24 more times</p>
<p style = "color: #000000; font-size: 16px;"> 5. 72 °C for 5 minutes</p>
<p style = "color: #000000; font-size: 16px;"> 6. 4 °C forever</p><br>
<p class = "serif" style = "color: #000000; font-size: 25px;"><span class = "mw-headline">Alpha-Thrombin-bound NHS ester bead affinity column:</span></p>
<hr>
<p style = "color: #000000; font-size: 16px;"><u>1. Prepare columns by functionalizing Pierce® NHS-Activated Agarose Dry Resin with alpha-thrombin:</u></p>
<p style = "color: #000000; font-size: 16px;"> a. In one pot, measure out 5 mg Pierce® NHS-Activated Agarose Dry Resin per each column to </p>
<p style = "color: #000000; font-size: 16px;"> be prepared.</p>
<p style = "color: #000000; font-size: 16px;"> b. Dilute alpha-thrombin stock into a 1 mg/mL 0.1 M HEPES + 0.15 M NaCl buffered solution with 30 μL volume per each column to be prepared.</p>
<p style = "color: #000000; font-size: 16px;"> c. Combine entire diluted alpha-thrombin stock with weighed-out Pierce® NHS-Activated Agarose Dry Resin.</p>
<p style = "color: #000000; font-size: 16px;"> d. Mix end-over-end for one hour in a filter column.</p>
<p style = "color: #000000; font-size: 16px;"> e. Centrifuge at 1000 rfc for 1 minute. Save flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> f. Add 30 μL volume per column to be prepared of 0.1 M HEPES, 0.15 M NaCl buffer. Recentrifuge at 1000 rfc for 1 minute and save </p>
<p style = "color: #000000; font-size: 16px;"> flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> g. Add 30 μL per column to be prepared of Tris buffer at pH 8 to quench column and mix end-over-end for 15-20 minutes at room</p>
<p style = "color: #000000; font-size: 16px;"> temperature.</p>
<p style = "color: #000000; font-size: 16px;"> h. Centrifuge at 1000 rfc for 1 minute and save flowthrough.</p>
<p style = "color: #000000; font-size: 16px;"> i. Add 30 μL volume per column to be prepared of 0.1 M HEPES + 0.15 M NaCl + 0.05% NaN3 buffer to resuspend resin after </p>
<p style = "color: #000000; font-size: 16px;"> quenching.</p>
<p style = "color: #000000; font-size: 16px;"> j. Split mix into columns with 30 μL suspended beads each and store at 4 °C.</p>
<p style = "color: #000000; font-size: 16px;"><u>2. Run column assay:</u></p>
<p style = "color: #000000; font-size: 16px;"> a. Prepare a 1 μM loading solution of the desired sample in 20 μL of a 1x TAE + 0.1M KCl buffered solution.</p>
<p style = "color: #000000; font-size: 16px;"> b. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting. Centrifuge at 1000 rfc for 1 minute.</p>
<p style = "color: #000000; font-size: 16px;"> c. Add 20 μL loading solution to column. Mix end-over-end for 1 hour.</p>
<p style = "color: #000000; font-size: 16px;"> d. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p>
<p style = "color: #000000; font-size: 16px;"> e. Add 60 μL 1x TAE + 0.1M KCl to column, and mix by gently pipetting.</p>
<p style = "color: #000000; font-size: 16px;"> f. Centrifuge at 1000 rfc for 1 minute, and save flowthrough in an Eppendorf tube.</p>
<p style = "color: #000000; font-size: 16px;"> g. Repeat steps e-f 3 more times. </p>
<p style = "color: #000000; font-size: 16px;"> h. Add 60 μL 1x TAE + 0.1M KCl to column, mix by gently pipetting, and pipet the contents of the column into an Eppendorf tube for </p>
<p style = "color: #000000; font-size: 16px;"> storage.</p>
<p style = "color: #000000; font-size: 16px;">Atomic Force Microscopy was performed by Shuoxing Jiang, a member of the Hao Yan Lab, using the Dimension FastScan AFM (Bruker). 2μl of sample was deposited onto freshly cleaved mica and left to absorb for 2 minutes. 100μl of 1x TAE Mg was pipetted on top of the DNA sample and the sample was imaged in ScanAsyst in Fluid mode using a ScanAssyst Fluid+ tip.</p>
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