Biomod/2012/TU Dresden/Nanosaurs/Lab book: Difference between revisions
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Revision as of 23:02, 27 October 2012
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<div id="main_saurs" style="width:960px;"> <div class="content_list"> <a href="#recipes">Recipes</a> <a href="#protocols">Protocols</a> <a href="#downloads">Downloads</a> </div> <hr> <a id="recipes"><h2>Recipes</h2></a>
<h3>DNA origami recipes</h3> <table> <tr> <th colspan="4"><center>FB 8 (1x / 10x)</center></th> </tr> <tr> <td><b>Description<b></td> <td colspan="3">Folding Buffer 8mM MgCl2: Buffer to be added before DNA origami assembly to create the necessary surrounding conditions for correct folding and assembly.</td> </tr>
<tr> <td><b>Recipe</b></td> <td> </td> <td><b>1x</b></td> <td><b>10x</b></td> </tr> <tr> <td> </td> <td>1M MgCl2</td> <td>8µl</td> <td>80µl</td> </tr> <tr> <td> </td> <td>1M Tris</td> <td>5µl</td> <td>50µl</td> </tr> <tr> <td> </td> <td>0,5M EDTA</td> <td>2µl</td> <td>20µl</td> </tr> <tr> <td> </td> <td>H2O</td> <td>985µl</td> <td>850µl</td> </tr> <tr> <td> </td> <td> </td> <td>1ml</td> <td>1ml</td> </tr> </table>
<table>
<tr> <th colspan="3"><center>Origami Assembly</center></th> </tr>
<tr> <td><b>Description</b></td> <td colspan="2">Combines staple strands and scaffold in the necessary surrounding conditions. Staple strands have an excess of 7,5x in comparison to the scaffold to increase the chances of correct assembly.</td> </tr> <tr> <td><b>Recipe</b></td> <td>100nM p7560 single strand</td> <td>10µl</td> </tr> <tr> <td> </td> <td>FB 8 10x</td> <td>10µl</td> </tr> <tr> <td> </td> <td>Staple mix (≈500nM per oligo)</td> <td>14,8µl</td> </tr> <tr> <td> </td> <td>H2O</td> <td>65,2µl</td> </tr> <tr> <td> </td> <td> </td> <td>100µl</td> </tr> </table>
<table>
<tr><th colspan="6"><center>Staple mix</center></th></tr> <tr><td><b>Description</b></td><td colspan="5">The certain mix of different staples (oligonucleotides) that are needed to <br/> form different structures with their respective functions.</td></tr> <tr><td><b>Recipe</b></td><td><b>Staple type</b></td><td><b>Open</b></td><td><b>Closed guided</b></td><td><b>Closed not guided</b></td><td> </td></tr> <tr><td> </td><td>core</td><td>x</td><td>x</td><td>x</td><td> </td></tr> <tr><td> </td><td>core_guideadj</td><td>x</td><td> </td><td>x</td><td> </td></tr> <tr><td> </td><td>guide_adj</td><td> </td><td>x</td><td> </td><td> </td></tr> <tr><td> </td><td>guide</td><td> </td><td>x</td><td> </td><td> </td></tr> <tr><td> </td><td>apt_locks</td><td> </td><td>x</td><td>x</td><td> </td></tr> <tr><td> </td><td>anchor</td><td>x</td><td>x</td><td>x</td><td> </td></tr> <tr><td> </td><td>catcher</td><td>x</td><td>x</td><td>x</td><td> </td></tr> <tr><td> </td><td>locks_nohang</td><td>x</td><td> </td><td> </td><td>If only the part inside the DNA origami and no overhang of the locks is wanted.</td></tr> <tr><td> </td><td>anchor_nohang</td><td> </td><td> </td><td> </td><td>If anchor strands are not wanted.</td></tr> <tr><td> </td><td>catcher_nohang</td><td> </td><td> </td><td> </td><td>If catcher strands are not wanted.</td></tr> <tr><td> </td><td>edge_stab_45</td><td> </td><td> </td><td> </td><td>For labeling of the DNA origami.</td></tr></table>
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<h3>GUV recipes</h3>
<table>
<tr> <th colspan="2"><center>SLB buffer</center></th> </tr>
<tr> <td><b>Description</b></td> <td>SLB (supported lipid bilayer) buffer is used as the solution for all the vesicle experiments.</td> </tr> <tr> <td><b>Recipe</b></td> <td>10mM HEPES</td> </tr> <tr> <td></td> <td>150mM NaCl</td> </tr> <tr> <td></td> <td>8mM Mg<sup>2++</sup></td> </tr> </table> <table>
<tr> <th colspan="3"><center>DNA PAGE gel 12%</center></th> </tr>
<tr> <td><b>Recipe</b></td> <td>Polyacrylamid</td> <td>3ml</td> </tr> <tr> <td></td> <td>H<sub>2</sub>O</td> <td>5,894ml</td> </tr> <tr> <td></td> <td>TBE 10x</td> <td>1ml</td> </tr> <tr> <td></td> <td>APS</td> <td>100µl</td> </tr> <tr> <td></td> <td>TEMED</td> <td>6µl</td> </tr> </table>
<table> <tr> <th><center>Ingredient</center></th> <th><center>Stock Concentration</center></th> <th><center>Amount (µl)</center></th> </tr> <tr> <td>TdT Buffer</td> <td> </td> <td>12</td> </tr>
<tr> <td>CoCl2</td> <td> </td> <td>12</td> </tr> <tr> <td>Oligonucleotide</td> <td>100 µM</td> <td>3</td> </tr> <tr> <td>Alexa-dUTP conjugate/<br/>Biotin-ddUTP conjugate</td> <td>1mM</td> <td>3</td></tr> <tr> <td>Terminal Transferase</td> <td> </td> <td>3</td> </tr> <tr> <td>Water</td> <td> </td> <td>27</td> </tr> <tr> <td><b>Total</b></td> <td> </td> <td>60</td> </tr> <tr> <td><b>Procedure</b></td> <td colspan="2">1. Incubate at 370 C for 60 mins.</td> </tr> <tr> <td> </td> <td colspan="2">2. Heat inactivate at 700 C for 15 mins.</td> </tr> <tr> <td> </td> <td colspan="2">3. Cleanup with NucleoSpinExtract II kit.</td> </tr> <tr> <td> </td> <td colspan="2">4. Measure the resultant concentration using <a href="http://www.nanodrop.com/?gclid=CPT5qOzCorMCFYq-zAodFA4Axw">NanoDrop</a></td> </tr> </table>
<a id="protocols"><h2>Protocols</h2>
<h3>Giant unilamellar vesicles (GUVs) formation</h3>
<p>The method used to form the GUVs is “Vesicle Formation from emulsion”. This method consists in a controlled hydration of dried or nearly dried films of lipids deposited on a solid surface, in this case electrodes. </p>
<ol> <li>Preparation of lipids solution</li> <p>The lipid mixture contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) in a 9:1 proportion in 1 mg/ml concentration. The solution is prepared with the organic solvent chloroform.</p> <li>Lipids spreading</li> <p>6.5 µl of the lipid mixture is evenly spread in the surface of two little wires, the electrodes, which are previously cleaned by sonication cycles, washed with ethanol and dried.</p> <li>Solvent evaporation</li> <p>The lipid sample is left to dry over the electrodes inside an extractor for at least 15 min to evacuate the solvent of chloroform.</p> <li>Electroformation</li> <p>The electrodes are inserted in the lid of a plastic chamber that is filled with 3.5 ml of sucrose solution isomolar to the working buffer (HEPES, NaCl and w/o Mg2+). This solution hydrates the dry lipid sample to form the GUVs emulsion. The hydration is carried out in the presence of an electric field with a frequency of 10 Hz during 2 hours to electroformate the vesicles and of 2 Hz during 30 min to detached them from the electrodes.</p>
</ol> <div class="img_right img_link"><a rel="lightbox" href="http://www.openwetware.org/images/4/4b/GUVs_formation.png"><img src="http://www.openwetware.org/images/4/44/GUVs_formations_s.png"></a> <div class="descr">Representation of electroformation/ eletroswelling</div></div>
<h3>Large unilamellar vesicles (LUVs) formation</h3>
<p>The method employed consists of rehydratation and extrusion, whose detailed procedures are summarized as following:</p>
<ol> <li>Preparation of lipids solution</li> <p>The lipid mixture of 1 mg/ml concentration contains 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) with 0%-10% DOPS volume. The solution is prepared with the organic solvent chloroform.</p> <li>Evaporation</li> <p>25 µl of sample solution are distributed as evenly as possible in the interior surface of a crystal meanwhile it is blown-dry using nitrogen. To totally evacuate the solvent of chloroform, the vial is left during one hour inside a drier. </p> <li>Hydratation</li> <p>500 µl of the working buffer (HEPES, NaCl and w/o Mg2+) is used to hydrate the dry lipid.</p> <li>Incubation</li> <p>The vials are left for 2 hours of incubation; mix the sample using a vortexer.</p> <li>Freeze/thaw</li> <p>The sample is immersed in liquid nitrogen followed by boiling water for 5 cycles totally.</p> <li>Extrusion</li> <p>Assemble the membrane inside the extruder as it follows:</p> <ol type="a"> <li>The Teflon piece is wetted with buffer.</li> <li>The 2 pieces of cellulose membrane are placed in a support in the center of the Teflon piece.</li> <li>Put the PC membrane with a pore size of 50-100 µm on the taller piece of the holder.</li> <li>A drop of buffer is placed on top of the membrane.</li> <li>The 2 pieces of Teflon holder are placed together.</li> <li>The syringe is filled with the lipid solution, and extrude at least 11 times.</li> <li>The extruded sample from the acceptor syringe is withdrawn. The solution should look clearer than it was before extrusion.</li> </ol> <li>Cleaning</li> <p>The Teflon piece, o-ring and syringe should be rinsed with copious 2-propanol and deionized water after use. Otherwise the residues will contaminate the next sample.</p> </ol>
<h2>Aptamer Lock</h2>
<h3>Optimization Experiments</h3>
<ol> <li>Equi-molar quanities of unlabeled aptamer strands and Cy3 labeled aptamer locking strands were mixed to produce samples with 1 nM, 10 nM and 100 nM concentrations of Cy3 labeled strands.</li>
<li> All the samples were made in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)</li>
<li> The samples were then annealed in a PCR system and spectrophotometrically measured.</li> <li> Spectrophotometric measurements very done using 120 µl cuvettes in a spectrophotometer.</li> <li> The samples were excited at 510 nm and emission was measured at 564 nm.</li> </ol>
<h3>Spectrophotometric Measurements</h3>
<ol> <li> All the spectrophotometric samples were prepared in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)</li>
<li> A 10 nM concentration of the respective labeled/unlabeld lock/oligo was used</li> <li> When PDGF was used, it was always used in 10 times excess concentration than the oligos</li>
<li> When either of the blockers were used, they were used in a 10 times excess concentration than the oligos</li> <li> All samples were incubated at room temperature for 24 Hrs.</li> <li> Spectrophotometric measurements very done using 120 µl cuvettes in a spectrophotometer.</li> <li> All the samples were excited at 510 nm and emission was measured at 624 nm</li> </ol>
<h3>Gel Shift Assays</h3>
<ol> <li> All the gel samples were prepared in SLB buffer (<a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">Lab Book -> Recipes</a>)</li>
<li> ~70 ng of DNA concentration was maintained for each sample.</li>
<li> When PDGF was used, it was always used in 10 times excess concentration than the lock/oligos</li> <li> When either of the blockers were used, they were used in a 10 times excess concentration than the lock/oligos</li> <li> All samples were incubated at room temperature for 24 Hrs.</li> <li> Fermentas 6X Orange DNA Loading Dye was used with all the samples
<li> Fermentas O’RangeRuler 20 bp DNA Ladder was used</li>
<li> Invitrogen, 4-20% TBE PAGE Gels were used and electrophoresis was performed for 40 minutes at 200 V</li> </ol>
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<a id="downloads"><h2>Downloads</h2></a> <ul>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/OligoList_for_DNA_Origami.xlsx">Oligo List for DNA origami</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/shell_final.json">shell_final.json</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/shell_final_guidestrands.json">shell_final_guidedstrands.json</a>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/p7560_sequence_cadnano2.txt">p7560 scaffold sequence from cadnano2</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/Lab_Book_DNA_Origami_group.docx">Lab Book of DNA origami group</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/TEM%20guide.docx">TEM guide</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/Staining%20Protocol.docx">Staining Protocol</a></li>
<li><a href="http://biomod-dresden-2012.googlecode.com/svn/trunk/downloads/Agarose%20gel%20electrophoresis.docx">Agarose gel electrophoresis</a></li>
</li>
</ul>
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