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color: orange; | color: orange; | ||
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<body> | <body> | ||
− | <div class=" | + | <div class="color1"> |
− | <h5 style="text-align: center"> Lab Journals </h5> | + | <div class="img_menu"> |
− | + | <h5 style="text-align: center">Lab Journals </h5> | |
− | + | <center> | |
− | + | <div class="image"> | |
− | + | <a href="https://2016.igem.org/Team:Freiburg/NotebookCloning"> <img class="imga" src="https://static.igem.org/mediawiki/2016/1/14/T--Freiburg--CloningLab.png" alt="Smiley face"></a> | |
− | + | <a href="https://2016.igem.org/Team:Freiburg/NotebookSpores"> <img class="imgb" src="https://static.igem.org/mediawiki/2016/9/93/T--Freiburg--SporesLab.png" alt="Smiley face"></a> | |
− | + | <a href="https://2016.igem.org/Team:Freiburg/NotebookProteins"> <img class="imgc" src="https://static.igem.org/mediawiki/2016/2/23/T--Freiburg--ProteinsLab.png" alt="Smiley face"></a> | |
− | + | <a href="https://2016.igem.org/Team:Freiburg/NotebookTargeting"><img class="imgd" src="https://static.igem.org/mediawiki/2016/f/f4/T--Freiburg--TargetingLab.png" alt="Smiley face"></a> | |
− | + | <a href="https://2016.igem.org/Team:Freiburg/NotebookDelivery"><img class="imge" src="https://static.igem.org/mediawiki/2016/4/4b/T--Freiburg--DeliveryLab.png" alt="Smiley face"></a> | |
− | + | </div> | |
+ | </center> | ||
+ | </div> | ||
</div> | </div> | ||
<div class="color8"> | <div class="color8"> | ||
<div class="para_center_20"> | <div class="para_center_20"> | ||
− | < | + | <h5 class="sectionedit1">Group 4 - Adhesion</h5> |
− | + | <div class="tags"> | |
− | + | The targeting is one of the main requirements the spores have to fulfill. An adhesion-assay was conducted to verify the binding of the spores to a desired target. | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
+ | <!-- para_20--> | ||
</div> | </div> | ||
+ | <!-- color --> | ||
<div class="color2"> | <div class="color2"> | ||
<div class="para_20"> | <div class="para_20"> | ||
<h3><a name="section07042016" id="section07042016">07/04/2016</a></h3> | <h3><a name="section07042016" id="section07042016">07/04/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p> <strong | + | <p> <strong>Bacillus subtilis strains used:</strong> </p> |
<div class="table sectionedit2"> | <div class="table sectionedit2"> | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0"> | + | <td class="col0"><strong>Strain</strong></td> |
− | + | ||
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0 | + | <td class="col0">B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5)</td> |
− | + | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0 | + | <td class="col0">B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5)</td> |
− | + | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0 | + | <td class="col0">B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5)</td> |
− | + | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0 | + | <td class="col0">B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5)</td> |
− | + | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0 | + | <td class="col0">B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5)</td> |
− | + | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT2 TABLE [ | + | <!-- EDIT2 TABLE [128-427] --> |
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</div> | </div> | ||
<h3><a name="section07052016" id="section07052016">07/05/2016</a></h3> | <h3><a name="section07052016" id="section07052016">07/05/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p> <strong> | + | <p> <strong>Slide preparation: GOPTS</strong> </p> |
− | <p> Surface activation of glass slides by plasma generator, coated with GOPTS | + | <p> Surface activation of glass slides by plasma generator, coated with GOPTS 3-glycidyloxypropyl trimethoxysilane, washed with acetone and then stored for up to two weeeks. </p> |
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
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<h3><a name="section07102016" id="section07102016">07/10/2016</a></h3> | <h3><a name="section07102016" id="section07102016">07/10/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p> <strong>Buffer for anti-GFP | + | <p> <strong>Buffer for anti-GFP nanobody (10 ml)</strong> </p> |
<ul> | <ul> | ||
<li class="level1"> | <li class="level1"> | ||
Line 156: | Line 146: | ||
</div> | </div> | ||
</div> | </div> | ||
− | <!-- | + | <!-- para_20--> |
</div> | </div> | ||
<!-- color --> | <!-- color --> | ||
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<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with nanobody (0.5µl, 330µg/ml, overnight, 4°C) </div> |
− | + | ||
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</li> | </li> | ||
</ol> | </ol> | ||
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</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top </div> |
</li> | </li> | ||
</ol> | </ol> | ||
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<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B52</td> | <td class="col1">GOPTS + nanobody + B52</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody + B54</td> | <td class="col1">GOPTS + nanobody + B54</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + B55</td> | <td class="col1">GOPTS + nanobody + B55</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + nanobody + B56</td> | <td class="col1">GOPTS + nanobody + B56</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT3 TABLE [ | + | <!-- EDIT3 TABLE [1598-1758] --> |
− | + | ||
− | + | ||
</div> | </div> | ||
<h3><a name="section07142016" id="section07142016">07/14/2016</a></h3> | <h3><a name="section07142016" id="section07142016">07/14/2016</a></h3> | ||
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</ol> | </ol> | ||
<p> <strong>Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56</strong> </p> | <p> <strong>Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56</strong> </p> | ||
− | <p> –> | + | <p> –> pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls |
<br/> </p> | <br/> </p> | ||
<p> <strong>Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56</strong> | <p> <strong>Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56</strong> | ||
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<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B52</td> | <td class="col1">GOPTS + nanobody + B52</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody + B54</td> | <td class="col1">GOPTS + nanobody + B54</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + B55</td> | <td class="col1">GOPTS + nanobody + B55</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + nanobody + B56</td> | <td class="col1">GOPTS + nanobody + B56</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">GOPTS + GFP (positive control)</td> | <td class="col1">GOPTS + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">GOPTS + WT (negative control)</td> | <td class="col1">GOPTS + WT (negative control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT4 TABLE [ | + | <!-- EDIT4 TABLE [2287-2516] --> |
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</div> | </div> | ||
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<h3><a name="section07152016" id="section07152016">07/15/2016</a></h3> | <h3><a name="section07152016" id="section07152016">07/15/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
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</ol> | </ol> | ||
</div> | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color4"> | ||
+ | <div class="para_20"> | ||
<h3><a name="section0723016" id="section0723016">07/23/016</a></h3> | <h3><a name="section0723016" id="section0723016">07/23/016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p | + | <p> <strong>Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56</strong> </p> |
− | + | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> washed slides with ddH2O</div> |
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with B52, B53, B54, B55, B56 (50µl/spot, overnight)</div> |
− | + | ||
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</li> | </li> | ||
</ol> | </ol> | ||
+ | <p> –> <strong><em class="u">FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP</em></strong> </p> | ||
</div> | </div> | ||
<h3><a name="section07242016" id="section07242016">07/24/2016</a></h3> | <h3><a name="section07242016" id="section07242016">07/24/2016</a></h3> | ||
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<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
− | <td class="col1"> | + | <td class="col1">B52</td> |
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
− | <td class="col1"> | + | <td class="col1">B53</td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
− | <td class="col1"> | + | <td class="col1">B54</td> |
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT5 TABLE [ | + | <!-- EDIT5 TABLE [3152-3201] --> |
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<p> <strong>Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56</strong> </p> | <p> <strong>Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56</strong> </p> | ||
− | < | + | <p> –> fluorescence signal of B53 and B54 </p> |
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<p> <strong>Fluorescence microscopy 3 (Olympus 1×71): B53-B55</strong> </p> | <p> <strong>Fluorescence microscopy 3 (Olympus 1×71): B53-B55</strong> </p> | ||
+ | <p> –> fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS </p> | ||
+ | <p> <strong>Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d) </div> |
</li> | </li> | ||
</ol> | </ol> | ||
− | + | </div> | |
− | + | <h3><a name="section07262016" id="section07262016">07/26/2016</a></h3> | |
− | + | <div class="level4"> | |
<p> <strong>Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | <p> <strong>Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> blocked with BSA(4%) (overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
</div> | </div> | ||
</div> | </div> | ||
− | <!-- | + | <!-- para_20--> |
</div> | </div> | ||
<!-- color --> | <!-- color --> | ||
<div class="color5"> | <div class="color5"> | ||
<div class="para_20"> | <div class="para_20"> | ||
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<h3><a name="section07272016" id="section07272016">07/27/2016</a></h3> | <h3><a name="section07272016" id="section07272016">07/27/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
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</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl/spot, overnight)</div> |
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</li> | </li> | ||
</ol> | </ol> | ||
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<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + GFP (positive control)</td> | <td class="col1">GOPTS + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
− | <td class="col1"> | + | <td class="col1">GOPTS + nanobody + GFP</td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + BSA + GFP (negative control)</td> | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + B53 (positive control)</td> | <td class="col1">GOPTS + B53 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + B54 (positive control)</td> | <td class="col1">GOPTS + B54 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">B53 (positive control)</td> | <td class="col1">B53 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">B54 (positive control)</td> | <td class="col1">B54 (positive control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT6 TABLE [ | + | <!-- EDIT6 TABLE [3988-4240] --> |
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</div> | </div> | ||
<h3><a name="section07282016" id="section07282016">07/28/2016</a></h3> | <h3><a name="section07282016" id="section07282016">07/28/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
<p> <strong>Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | <p> <strong>Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
− | <p> –>spores appear to not have bound to nanobody | + | <p> –> spores appear to not have been bound to nanobody </p> |
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</div> | </div> | ||
<h3><a name="section08082016" id="section08082016">08/08/2016</a></h3> | <h3><a name="section08082016" id="section08082016">08/08/2016</a></h3> | ||
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</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl/spot, overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
Line 594: | Line 469: | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B55 (negative control)</td> | <td class="col1">GOPTS + nanobody + B55 (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + B55 (negative control)</td> | <td class="col1">GOPTS + B55 (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + GFP (positive control)</td> | <td class="col1">GOPTS + nanobody + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + BSA + GFP (negative control)</td> | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">B53 (positive control)</td> | <td class="col1">B53 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">B54 (positive control)</td> | <td class="col1">B54 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row8"> | <tr class="row8"> | ||
− | <td class="col0">8 | + | <td class="col0">8</td> |
<td class="col1">GOPTS + GFP (positive control)</td> | <td class="col1">GOPTS + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row9"> | <tr class="row9"> | ||
− | <td class="col0">9 | + | <td class="col0">9</td> |
<td class="col1">GOPTS + B53 (positive control)</td> | <td class="col1">GOPTS + B53 (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row10"> | <tr class="row10"> | ||
− | <td class="col0">10 | + | <td class="col0">10</td> |
<td class="col1">GOPTS + nanobody(Alexa647) + B53</td> | <td class="col1">GOPTS + nanobody(Alexa647) + B53</td> | ||
</tr> | </tr> | ||
<tr class="row11"> | <tr class="row11"> | ||
− | <td class="col0">11 | + | <td class="col0">11</td> |
<td class="col1">GOPTS + nanobody(Alexa647) + B54</td> | <td class="col1">GOPTS + nanobody(Alexa647) + B54</td> | ||
</tr> | </tr> | ||
<tr class="row12"> | <tr class="row12"> | ||
− | <td class="col0">12 | + | <td class="col0">12</td> |
<td class="col1">GOPTS + nanobody(Alexa647) + WT(negative control)</td> | <td class="col1">GOPTS + nanobody(Alexa647) + WT(negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row13"> | <tr class="row13"> | ||
− | <td class="col0">13 | + | <td class="col0">13</td> |
<td class="col1">GOPTS + nanobody(Alexa647) + B56 (negative control)</td> | <td class="col1">GOPTS + nanobody(Alexa647) + B56 (negative control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT7 TABLE [ | + | <!-- EDIT7 TABLE [4840-5372] --> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
− | <!-- | + | <!-- para_20--> |
</div> | </div> | ||
<!-- color --> | <!-- color --> | ||
− | <div class=" | + | <div class="color6"> |
<div class="para_20"> | <div class="para_20"> | ||
+ | <h3><a name="section08092016" id="section08092016">08/09/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP</strong> </p> | ||
+ | <p> –> spores have not been bound </p> | ||
+ | </div> | ||
<h3><a name="section08102016" id="section08102016">08/10/2016</a></h3> | <h3><a name="section08102016" id="section08102016">08/10/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
Line 719: | Line 565: | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">nanobody + WT (negative control)</td> | <td class="col1">nanobody + WT (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">nanobody + B53</td> | <td class="col1">nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">nanobody + B53</td> | <td class="col1">nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">nanobody + B55 (negative control)</td> | <td class="col1">nanobody + B55 (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">nanobody + B56(negative control)</td> | <td class="col1">nanobody + B56(negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">nanobody + GFP (positive control)</td> | <td class="col1">nanobody + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">nanobody (positive control)</td> | <td class="col1">nanobody (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row8"> | <tr class="row8"> | ||
− | <td class="col0">8 | + | <td class="col0">8</td> |
<td class="col1">GFP (positive control)</td> | <td class="col1">GFP (positive control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT8 TABLE [ | + | <!-- EDIT8 TABLE [5936-6208] --> |
− | + | ||
− | + | ||
<p> <strong>Fluorescence microscopy 6 (Nikon C2+ confocal microscope): anti-GFP nanobody + spores (WT, B53, B54, B56) in solution</strong> </p> | <p> <strong>Fluorescence microscopy 6 (Nikon C2+ confocal microscope): anti-GFP nanobody + spores (WT, B53, B54, B56) in solution</strong> </p> | ||
<p> –> no introduction to confocal mode: nanobody not observable </p> | <p> –> no introduction to confocal mode: nanobody not observable </p> | ||
− | |||
</div> | </div> | ||
<h3><a name="section08142016" id="section08142016">08/14/2016</a></h3> | <h3><a name="section08142016" id="section08142016">08/14/2016</a></h3> | ||
Line 795: | Line 638: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
Line 801: | Line 644: | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">nanobody + WT (negative control)</td> | <td class="col1">nanobody + WT (negative control)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">nanobody + B53</td> | <td class="col1">nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">nanobody + B54</td> | <td class="col1">nanobody + B54</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">nanobody + GFP (positive control)</td> | <td class="col1">nanobody + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">nanobody (positive control)</td> | <td class="col1">nanobody (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">GFP (positive control)</td> | <td class="col1">GFP (positive control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT9 TABLE [ | + | <!-- EDIT9 TABLE [6899-7096] --> |
− | + | <p> <strong> Washing test A: GOPTS + anti-GFP nanobody + B53</strong> </p> | |
− | + | ||
− | <p> <strong>Washing test:</strong> | + | |
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)</div> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</li> | </li> | ||
</ol> | </ol> | ||
</div> | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color7"> | ||
+ | <div class="para_20"> | ||
<h3><a name="section08152016" id="section08152016">08/15/2016</a></h3> | <h3><a name="section08152016" id="section08152016">08/15/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong | + | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> </p> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
Line 874: | Line 704: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with B53 (10µl/spot, overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
− | |||
− | |||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<h3><a name="section08162016" id="section08162016">08/16/2016</a></h3> | <h3><a name="section08162016" id="section08162016">08/16/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
<p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> </p> | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> </p> | ||
− | < | + | <ol> |
− | < | + | <li class="level1"> |
− | < | + | <div class="li"> rinsed and washed (see table below)</div> |
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddh2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
<div class="table sectionedit10"> | <div class="table sectionedit10"> | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
− | <td class="col2">Condition</td> | + | <td class="col2"><strong>Condition</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed 2x with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed 2x with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed 3x with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed 3x with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed 4x with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed 4x with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm)</td> | <td class="col2">washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm)</td> | <td class="col2">washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm)</td> | ||
Line 926: | Line 759: | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT10 TABLE [ | + | <!-- EDIT10 TABLE [7723-8129] --> |
− | + | ||
− | + | ||
</div> | </div> | ||
− | <h3><a name=" | + | <h3><a name="section08172016" id="section08172016">08/17/2016</a></h3> |
<div class="level4"> | <div class="level4"> | ||
<p> <strong>Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | <p> <strong>Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
− | <p> –> spores have not bound to nanobody | + | <p> –> spores have not bound to nanobody </p> |
− | + | <p> <strong>Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53</strong> </p> | |
− | <p> <strong>Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53</strong> –>spores have not bound to BSA | + | <p> –> spores have not bound to BSA </p> |
− | + | ||
</div> | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color8"> | ||
+ | <div class="para_20"> | ||
<h3><a name="section08212016" id="section08212016">08/21/2016</a></h3> | <h3><a name="section08212016" id="section08212016">08/21/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
<p> <strong>Sample preparation 8: GOPTS + anti-GFP nanobody + GFP</strong> </p> | <p> <strong>Sample preparation 8: GOPTS + anti-GFP nanobody + GFP</strong> </p> | ||
− | |||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with nanobody (see table below, 60 min)</div> |
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
Line 971: | Line 806: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl, overnight) |
<br/> </div> | <br/> </div> | ||
</li> | </li> | ||
Line 978: | Line 813: | ||
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + GFP (1µl) (positive control)</td> | <td class="col1">GOPTS + GFP (1µl) (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + GFP (5 µl) (positive control)</td> | <td class="col1">GOPTS + GFP (5 µl) (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody (1µl, 330µg/ml) + GFP</td> | <td class="col1">GOPTS + nanobody (1µl, 330µg/ml) + GFP</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody (0.5µl, 10mg/ml) + GFP</td> | <td class="col1">GOPTS + nanobody (0.5µl, 10mg/ml) + GFP</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5, 6</td> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<td class="col1">GOPTS + BSA + GFP (negative control)</td> | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- EDIT11 TABLE [ | + | <!-- EDIT11 TABLE [8932-9176] --> |
− | + | ||
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− | + | ||
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− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<h3><a name="section08222016" id="section08222016">08/22/2016</a></h3> | <h3><a name="section08222016" id="section08222016">08/22/2016</a></h3> | ||
<div class="level4"> | <div class="level4"> | ||
− | <p> <strong>Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP</strong> | + | <p> <strong>Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP</strong> </p> |
− | + | <p> –> GFP not bound, GOPTS plates too old </p> | |
</div> | </div> | ||
<h3><a name="section08232016" id="section08232016">08/23/2016</a></h3> | <h3><a name="section08232016" id="section08232016">08/23/2016</a></h3> | ||
Line 1,080: | Line 850: | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with GFP (1.35mg/ml, volume/incubation: see table below)</div> |
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
Line 1,095: | Line 865: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
− | <div class="table | + | <div class="table sectionedit12"> |
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
− | <td class="col1">GOPTS + GFP (1 µl, 10 min) </td> | + | <td class="col1">GOPTS + GFP (1 µl, 10 min)</td> |
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
− | <td class="col1">GOPTS + GFP (2 µl, 10 min) </td> | + | <td class="col1">GOPTS + GFP (2 µl, 10 min)</td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + GFP (5 µl, 10 min)</td> | <td class="col1">GOPTS + GFP (5 µl, 10 min)</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + GFP (1 µl, 15 min)</td> | <td class="col1">GOPTS + GFP (1 µl, 15 min)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + GFP (2 µl, 15 min)</td> | <td class="col1">GOPTS + GFP (2 µl, 15 min)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">GOPTS + GFP (5 µl, 15 min)</td> | <td class="col1">GOPTS + GFP (5 µl, 15 min)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">GOPTS + GFP (1 µl, 20 min)</td> | <td class="col1">GOPTS + GFP (1 µl, 20 min)</td> | ||
</tr> | </tr> | ||
<tr class="row8"> | <tr class="row8"> | ||
− | <td class="col0">8 | + | <td class="col0">8</td> |
<td class="col1">GOPTS + GFP (2 µl, 20 min)</td> | <td class="col1">GOPTS + GFP (2 µl, 20 min)</td> | ||
</tr> | </tr> | ||
<tr class="row9"> | <tr class="row9"> | ||
− | <td class="col0">9 | + | <td class="col0">9</td> |
<td class="col1">GOPTS + GFP (5 µl, 20 min)</td> | <td class="col1">GOPTS + GFP (5 µl, 20 min)</td> | ||
</tr> | </tr> | ||
<tr class="row10"> | <tr class="row10"> | ||
− | <td class="col0">10 | + | <td class="col0">10</td> |
<td class="col1">GOPTS (negative control)</td> | <td class="col1">GOPTS (negative control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- | + | <!-- EDIT12 TABLE [9664-10007] --> |
− | <p | + | <p> <strong>Sample preparation 10: GOPTS + anti-GFP nanobody + B53</strong> </p> |
− | + | ||
− | + | ||
<ol> | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with nanobody (1µl, 330µg/ml, 60 min)</div> | ||
+ | </li> | ||
<li class="level1"> | <li class="level1"> | ||
<div class="li"> rinsed and blocked with BSA(4%) (60 min)</div> | <div class="li"> rinsed and blocked with BSA(4%) (60 min)</div> | ||
Line 1,176: | Line 947: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> |
− | + | ||
</li> | </li> | ||
</ol> | </ol> | ||
− | + | <div class="table sectionedit13"> | |
− | + | ||
− | <div class="table | + | |
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + GFP(positive control)</td> | <td class="col1">GOPTS + GFP(positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + GFP (positive control)</td> | <td class="col1">GOPTS + nanobody + GFP (positive control)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + BSA + B53 (negative control)</td> | <td class="col1">GOPTS + BSA + B53 (negative control)</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- | + | <!-- EDIT13 TABLE [10477-10677] --> |
− | <p | + | <p> <strong>Washing test B: GOPTS + anti-GFP nanobody + B53</strong> </p> |
− | + | ||
<ol> | <ol> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with nanobody (1µl, 330µg/ml, 60 min)</div> |
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
Line 1,242: | Line 1,009: | ||
</li> | </li> | ||
<li class="level1"> | <li class="level1"> | ||
− | <div class="li"> | + | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> |
</li> | </li> | ||
</ol> | </ol> | ||
− | + | <div class="table sectionedit14"> | |
− | + | ||
− | <div class="table | + | |
<table class="inline"> | <table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
− | <td class="col0">Spot | + | <td class="col0"><strong>Spot No.</strong></td> |
− | <td class="col1">Sample</td> | + | <td class="col1"><strong>Sample</strong></td> |
− | <td class="col2">Condition</td> | + | <td class="col2"><strong>Condition</strong></td> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
− | <td class="col0">1 | + | <td class="col0">1</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">rinsed with PBS(1x) </td> | <td class="col2">rinsed with PBS(1x) </td> | ||
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
− | <td class="col0">2 | + | <td class="col0">2</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">swivel with PBS(1x) (0.5 min)</td> | <td class="col2">swivel with PBS(1x) (0.5 min)</td> | ||
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
− | <td class="col0">3 | + | <td class="col0">3</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed with PBS(1x) (1 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (1 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
− | <td class="col0">4 | + | <td class="col0">4</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed with PBS(1x) (2 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (2 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
− | <td class="col0">5 | + | <td class="col0">5</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed with PBS(1x) (3 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (3 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row6"> | <tr class="row6"> | ||
− | <td class="col0">6 | + | <td class="col0">6</td> |
<td class="col1">GOPTS + nanobody + B53</td> | <td class="col1">GOPTS + nanobody + B53</td> | ||
<td class="col2">washed with PBS(1x) (5 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (5 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row7"> | <tr class="row7"> | ||
− | <td class="col0">7 | + | <td class="col0">7</td> |
<td class="col1">GOPTS + BSA + B53(negative control)</td> | <td class="col1">GOPTS + BSA + B53(negative control)</td> | ||
<td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row8"> | <tr class="row8"> | ||
− | <td class="col0">8 | + | <td class="col0">8</td> |
<td class="col1">GOPTS + BSA + GFP(negative control)</td> | <td class="col1">GOPTS + BSA + GFP(negative control)</td> | ||
<td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row9"> | <tr class="row9"> | ||
− | <td class="col0">9 | + | <td class="col0">9</td> |
<td class="col1">GOPTS + GFP (positive control)</td> | <td class="col1">GOPTS + GFP (positive control)</td> | ||
<td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
</tr> | </tr> | ||
<tr class="row10"> | <tr class="row10"> | ||
− | <td class="col0">10 | + | <td class="col0">10</td> |
<td class="col1">GOPTS + B53 (positive control)</td> | <td class="col1">GOPTS + B53 (positive control)</td> | ||
<td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
Line 1,306: | Line 1,071: | ||
</table> | </table> | ||
</div> | </div> | ||
− | <!-- | + | <!-- EDIT14 TABLE [11141-11836] --> |
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color1"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section08242016" id="section08242016">08/24/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 9 (Nikon C2+ confocal microscope): GOPTS + GFP</strong> </p> | ||
+ | <p> –> strong fluorescence signal at some of the edges: incubation too long/volume too little, GFP has dried and could not have been washed away properly </p> | ||
<p> | <p> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/5/5c/T--Freiburg--targetingjournal1.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/87/T--Freiburg--targetingjournal2.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/2/25/T--Freiburg--targetingjournal3.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/6/66/T--Freiburg--targetingjournal4.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/4/46/T--Freiburg--targetingjournal5.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/00/T--Freiburg--targetingjournal6.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/af/T--Freiburg--targetingjournal7.png"> </center> | ||
+ | |||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 1) GOPTS + GFP (1 µl, 10 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 2) GOPTS + GFP (2 µl, 10 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 3) GOPTS + GFP (5 µl, 10 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 4) GOPTS + GFP (1 µl, 15 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 5) GOPTS + GFP (2 µl, 15 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 6) GOPTS + GFP (5 µl, 15 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 7) GOPTS + GFP (1 µl, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 8) GOPTS + GFP (2 µl, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 9) GOPTS + GFP (5 µl, 20 min)</div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p> <strong>Fluorescence microscopy 10 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53</strong> </p> | ||
+ | <p> –> no spores appear to have bound to nanobody </p> | ||
+ | <p> <strong>Fluorescence microscopy B (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53</strong> </p> | ||
+ | <p> –> no spores after washing, no binding between nanobody and B53? </p> | ||
+ | </div> | ||
+ | <h3><a name="section08252016" id="section08252016">08/25/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Blocking test C: GOPTS + BSA/milk powder + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> blocking with BSA(4%)/milk powder(5%) (120 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with GFP (1µl/spot, 1.35mg/ml, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH20</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit15"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1</td> | ||
+ | <td class="col1">GOPTS + GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">2</td> | ||
+ | <td class="col1">GOPTS + milk powder(5%) + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">GOPTS + BSA(4%) + GFP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT15 TABLE [13676-13797] --> | ||
+ | <p> <strong>Contamination Test D: BSA, PBS Mowiol DABCO</strong> </p> | ||
+ | <p> –> no contamination/pollution detected </p> | ||
+ | <p> <strong>Sample preparation 11: GOPTS + mCherry</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with mCherry (1µl, 2mg/ml, incubation: see table below)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with BSA(4%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit16"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1</td> | ||
+ | <td class="col1">GOPTS + mCherry (5 min) </td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">2</td> | ||
+ | <td class="col1">GOPTS + mCherry (10 min)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">GOPTS + mCherry (15 min)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">4</td> | ||
+ | <td class="col1">GOPTS (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">mCherry + Mowiol DABCO (positive control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT16 TABLE [14206-14394] --> | ||
+ | <p> <strong>Sample preparation 12: GOPTS + anti-GFP nanobody + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with nanobody (5µl, 330µg/ml, 120 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (2µl, 1.35mg/ml, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH20</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit17"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1</td> | ||
+ | <td class="col1">GOPTS + nanobody + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">2</td> | ||
+ | <td class="col1">GOPTS (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">GOPTS + GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">4</td> | ||
+ | <td class="col1">GOPTS + nanobody (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT17 TABLE [14873-15071] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color2"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section08272016" id="section08272016">08/27/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy C (Nikon C2+ confocal microscope): Blocking test: GOPTS + BSA/milkpowder + GFP</strong> </p> | ||
+ | <p> –> No GFP signal after blocking with BSA and milk powder </p> | ||
+ | <p> <strong>Fluorescence microscopy 11 (Nikon C2+ confocal microscope): GOPTS + mCherry</strong> </p> | ||
+ | <p> –> no mCherry-signal, too weak (quantum yield of mCherry about 1/3 of that of GFP) </p> | ||
+ | <p> <strong>Fluorescence microscopy 12 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP</strong> </p> | ||
+ | <p> –> GFP has not bound to nanobody </p> | ||
+ | </div> | ||
+ | <h3><a name="section08302016" id="section08302016">08/30/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 13: GOPTS + nanobody + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with nanobody (5µl, 330µg/ml, 60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (2µl, 1.35mg/ml, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH20</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit18"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1,2</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3,4</td> | ||
+ | <td class="col1">GOPTS + nanobody + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">GOPTS + GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">6</td> | ||
+ | <td class="col1">GOPTS + nanobody (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">7</td> | ||
+ | <td class="col1">GOPTS + BSA (negative control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT18 TABLE [16042-16246] --> | ||
+ | </div> | ||
+ | <h3><a name="section09012016" id="section09012016">09/01/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 13 (Nikon C2+ confocal microscope): GOPTS + nanobody + GFP</strong> </p> | ||
+ | <p> –> no GFP signal detectable </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color3"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section09062016" id="section09062016">09/06/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 14: GOPTS + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (10µl, 1.35mg/ml, 30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit19"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1,2</td> | ||
+ | <td class="col1">GOPTS + mCherry</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">GOPTS + BSA + mCherry (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">4</td> | ||
+ | <td class="col1">GOPTS + BSA (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">mCherry (positive control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT19 TABLE [16703-16861] --> | ||
+ | <p> <em class="u">Mowiol was produced due to this protocol:</em> <a href="https://static.igem.org/mediawiki/2016/5/56/T--Freiburg--LabJournal98.pdf" class="urlextern" target="_Blank" title="https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf" rel="nofollow">https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf</a> | ||
<br/> </p> | <br/> </p> | ||
+ | </div> | ||
+ | <h3><a name="section09072016" id="section09072016">09/07/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 14 (Nikon C2+ confocal microscope): GOPTS + GFP</strong> </p> | ||
+ | <p> –> spots with evenly distributed GFP </p> | ||
+ | <p> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/c/cc/T--Freiburg--targetingjournal8.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/a1/T--Freiburg--targetingjournal9.png"> </center> | ||
+ | |||
+ | <br/> </p> | ||
+ | <ul> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 1) GOPTS + GFP</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 2) GOPTS + nanobody + GFP</div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p> | ||
+ | <br/> </p> | ||
+ | </div> | ||
+ | <h3><a name="section09142016" id="section09142016">09/14/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 16: GOPTS + B53</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with B53 (5µl, 60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(4%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit20"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1,2,3</td> | ||
+ | <td class="col1">GOPTS + B53</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">4</td> | ||
+ | <td class="col1">GOPTS + WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">6</td> | ||
+ | <td class="col1">B53 (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">7</td> | ||
+ | <td class="col1">GOPTS + GFP</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT20 TABLE [17589-17737] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color4"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section09152016" id="section09152016">09/15/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 17: GOPTS + anti-GFP nanobody(Alexa647) + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with nanobody (5µl, 330µg/ml, 60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (2µl, 1.35mg/ml, 20 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH20</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit21"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1-4</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">GOPTS + milk powder + GFP (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">6</td> | ||
+ | <td class="col1">GOPTS + milk powder + NB (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7</td> | ||
+ | <td class="col1">GOPTS + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">8</td> | ||
+ | <td class="col1">GOPTS (negative control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT21 TABLE [18244-18450] --> | ||
+ | <p> <strong>Fluorescence microscopy 16 (Nikon C2+ confocal microscope): GOPTS + B53</strong> </p> | ||
+ | <p> –> no spores detectable </p> | ||
+ | </div> | ||
+ | <h3><a name="section09162016" id="section09162016">09/16/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 17 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP</strong> </p> | ||
+ | <p> –> GFP signal: GFP has been bound to nanobody </p> | ||
+ | <p> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/0/02/T--Freiburg--targetingjournal10.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/8c/T--Freiburg--targetingjournal11.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/83/T--Freiburg--targetingjournal20.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/a9/T--Freiburg--targetingjournal13.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/b6/T--Freiburg--targetingjournal14.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/2/25/T--Freiburg--targetingjournal15.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/e/e4/T--Freiburg--targetingjournal16.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/4/4b/T--Freiburg--targetingjournal17.png"> </center> | ||
+ | |||
+ | <br/> </p> | ||
+ | <ul> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 1-4) GOPTS + nanobody(Alexa647) + GFP</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 5) GOPTS + milk powder + GFP (negative control)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 6) GOPTS + milk powder + NB (negative control)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 7) GOPTS + GFP (positive control)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 8) GOPTS (negative control)</div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> </p> | ||
+ | </div> | ||
+ | <h3><a name="section09172016" id="section09172016">09/17/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 18: GOPTS + anti-GFP nanobody + GFP (spotting-mask)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with anti-GFP nanobody ( 2µl/spot, 330µg/ml, 10 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsing and blocking with milk powder(5%) (15 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsing with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> (removed mask)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color5"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section09182016" id="section09182016">09/18/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 18 (Nikon C2+ confocal microscope): GOPTS + antiGFP-nanobody + GFP (spotting-mask)</strong> </p> | ||
+ | <p> –> no GFP signal detectable </p> | ||
+ | <p> <strong>Sample preparation 19: GOPTS + anti-GFP nanobody + GFP (spotting-mask)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with anti-GFP nanobody (2µl/spot, 330µg/ml, 15 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsing with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> (removed mask)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsing and blocking with milk powder(5%) (15 min, 2 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> (reattached mask)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> (removed mask)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <h3><a name="section09192016" id="section09192016">09/19/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 19 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP (spotting-mask)</strong> </p> | ||
+ | <p> –> slight GFP-signal on nanobody </p> | ||
+ | <p> <strong>Sample preparation 20: anti-GFP nanobody + B53 (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 15 min, 4°C) + B53</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (2µl, 15 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p> –> problem: no washing away of unbound nanobody </p> | ||
+ | <div class="table sectionedit22"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1,2</td> | ||
+ | <td class="col1">B53 + nanobody(Alexa647) (incubated in solution) on GOPTS</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3,4</td> | ||
+ | <td class="col1">WT + nanobody(Alexa647) (incubated in solution) on GOPTS (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5,6</td> | ||
+ | <td class="col1">B53 + nanobody(Alexa647) (incubated in solution) dried on milk powder</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7,8</td> | ||
+ | <td class="col1">WT + nanobody(Alexa647) (incubated in solution) dried on milk powder (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9,10</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) + GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11,12</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) + B53</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13,14</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) + WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15</td> | ||
+ | <td class="col1">GOPTS + milk powder + nanobody(Alexa647) (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">16</td> | ||
+ | <td class="col1">GOPTS + milk powder + B53 (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">17</td> | ||
+ | <td class="col1">GOPTS + milk powder + WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">18</td> | ||
+ | <td class="col1">GOPTS + GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">19</td> | ||
+ | <td class="col1">GOPTS + nanobody(Alexa647) (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">20</td> | ||
+ | <td class="col1">B53 dried (positive control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT22 TABLE [21203-21988] --> | ||
+ | </div> | ||
+ | <h3><a name="section09202016" id="section09202016">09/20/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 21: anti-GFP nanobody + B53 (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 30 min, 4°C) + B53</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> centrifuged (2 min, 13400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> repeated steps 2 and 3 </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (30µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (2µl, 15 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milkpowder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit23"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1</td> | ||
+ | <td class="col1">B53 + nanobody(Alexa647) (incubated in solution) on GOPTS</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">2</td> | ||
+ | <td class="col1">WT + nanobody(Alexa647) (incubated in solution) on GOPTS</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">3,4</td> | ||
+ | <td class="col1">B53 + nanobody(Alexa647) dried on milkpowder</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">5,6</td> | ||
+ | <td class="col1">WT + nanobody(Alexa647) dried on milkpowder</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT23 TABLE [22443-22692] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color6"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section09292016" id="section09292016">09/29/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 22: GOPTS + GFP</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with GFP (1 µl, 1.35mg/ml, 30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (5µl, overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit24"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0 leftalign">1-5 </td> | ||
+ | <td class="col1">GOPTS + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0 leftalign">6 </td> | ||
+ | <td class="col1">GOPTS</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT24 TABLE [23013-23068] --> | ||
+ | </div> | ||
+ | <h3><a name="section09302016" id="section09302016">09/30/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <em class="u"><strong>–> Construct spores are ready for analysis</strong></em> </p> | ||
+ | <div class="table sectionedit25"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Strain No.</strong></td> | ||
+ | <td class="col1"><strong>Strain</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">150: aGFPnano_HA_aHelix_cgeA</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">6</td> | ||
+ | <td class="col1">151: aGFPnano_HA_aHelix_cotG</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">7</td> | ||
+ | <td class="col1">159: aGFPnano_HA_G4S_CotZ</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">9</td> | ||
+ | <td class="col1">157: aGFPnano_HA_aHelix_CotZ</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT25 TABLE [23141-23297] --> | ||
+ | <p> <strong>Fluorescence microscopy 22 (Nikon C2+ confocal microscope): GOPTS + GFP</strong> </p> | ||
+ | <p> –> spots with evenly distributed GFP </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with GFP (1 µl, 1.35mg/ml, 30 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> spores centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> step 1 repeated</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide(GFP) with spores (4µl, 400,000/µl, incubation time: see table below, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit26"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Incubation time (min)</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 3</td> | ||
+ | <td class="col2">5</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">2</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 3</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">3</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 3</td> | ||
+ | <td class="col2">15</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">4</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 6</td> | ||
+ | <td class="col2">5</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">5</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 6</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">6</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 6</td> | ||
+ | <td class="col2">15</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">7</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 7</td> | ||
+ | <td class="col2">5</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">8</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 7</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">9</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 7</td> | ||
+ | <td class="col2">15</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">10</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 9</td> | ||
+ | <td class="col2">5</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">11</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 9</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">12</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 9</td> | ||
+ | <td class="col2">15</td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GOPTS + GFP (positive control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">15</td> | ||
+ | <td class="col1">GOPTS + GFP + WT (negative control)</td> | ||
+ | <td class="col2">5</td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">16</td> | ||
+ | <td class="col1">GOPTS + GFP + WT (negative control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">17</td> | ||
+ | <td class="col1">GOPTS + GFP + WT (negative control)</td> | ||
+ | <td class="col2">15</td> | ||
+ | </tr> | ||
+ | <tr class="row17"> | ||
+ | <td class="col0">18</td> | ||
+ | <td class="col1">GOPTS + strain 3 (positive control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row18"> | ||
+ | <td class="col0">19</td> | ||
+ | <td class="col1">GOPTS + strain 6 (positive control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row19"> | ||
+ | <td class="col0">20</td> | ||
+ | <td class="col1">GOPTS + strain 7 (positive control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row20"> | ||
+ | <td class="col0">21</td> | ||
+ | <td class="col1">GOPTS + strain 9 (positive control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | <tr class="row21"> | ||
+ | <td class="col0">22</td> | ||
+ | <td class="col1">GOPTS + WT (negative control)</td> | ||
+ | <td class="col2">10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT26 TABLE [23872-24670] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color7"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section10012016" id="section10012016">10/01/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 23: GOPTS + GFP + spores</strong> </p> | ||
+ | <p> –> no spores detectable </p> | ||
+ | <p> <strong>Sample preparation 24: GOPTS + GFP + spores (in EtOH resuspended)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (10 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (4 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> step 2 repeated</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in EtOH (250µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide (4µl, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit27"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1-3</td> | ||
+ | <td class="col1">GOPTS + GFP + strain 6</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">4-6</td> | ||
+ | <td class="col1">GOPTS + GFP + WT</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">7-9</td> | ||
+ | <td class="col1">GOPTS + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">10-12</td> | ||
+ | <td class="col1">GOPTS + strain 6</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">13-15</td> | ||
+ | <td class="col1">GOPTS + WT</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT27 TABLE [25275-25414] --> | ||
+ | </div> | ||
+ | <h3><a name="section10022016" id="section10022016">10/02/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (strains 3,6,7,9,wt) (in EtOH resuspended)</strong> </p> | ||
+ | <p> –> EtOH denaturated GFP: very weak signal. No spores detectable </p> | ||
+ | <p> <strong>Sample preparation 25: GFP + spores (strains 3,6,7,9) (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (4µl, 1,35g/ml) + spores(strain 3,6,7,9) (4µl, 400,000/µl) (incubation time: see table below, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> centrifuged (2 min, 13400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> repeated steps 2 and 3 </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (20µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (4µl, 10 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubrnight)ation with Mowiol DABCO </div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit28"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + PBS (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">18</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">19</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">20</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT28 TABLE [26200-26582] --> | ||
+ | </div> | ||
+ | <h3><a name="section10032016" id="section10032016">10/03/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 26: Incubation test: GFP + spores (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (4µl, 1,35g/ml) + spores (4µl, 400,000/µl) (incubation time: see tables below, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> centrifuged (2 min, 13400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> repeated steps 2 and 3)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (20µl))</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (4µl, 10 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p> Slide 1: </p> | ||
+ | <div class="table sectionedit29"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Incubation time</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 3(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 6(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 7(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 9 (pellet washed withn PBS(1x))</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control) (pellet washed with PBS(1x))</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">26</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">27</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">28</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT29 TABLE [27172-27869] --> | ||
+ | <p> Slide 2: </p> | ||
+ | <div class="table sectionedit30"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Incubation time</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 3(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 6(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 7(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 9 (pellet washed withn PBS(1x))</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control) (pellet washed with PBS(1x))</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">26</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">27</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">28</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT30 TABLE [27881-28578] --> | ||
+ | <p> Slide 3: </p> | ||
+ | <div class="table sectionedit31"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Incubation time</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 3(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 6(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 7(pellet washed with PBS(1x))</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 9 (pellet washed withn PBS(1x))</td> | ||
+ | <td class="col2">120 min</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control) (pellet washed with PBS(1x))</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">26</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">27</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">28</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT31 TABLE [28590-29295] --> | ||
+ | <p> <strong>Sample preparation 28: GFP-volume test B: GFP + strain 7 (in solution, washed)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (volume: see table below, 1,35g/ml) + strain 7 (4µl, 400,000/µl) (90 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> centrifuged (2 min, 13400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> repeated steps 2 and 3)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (20µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (2µl, 90 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit32"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Volume (GFP)</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">0,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">1,0µl</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">1,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">2,0µl</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">2,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2">4,0µl</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">0,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">1,0µl</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">1,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">2,0µl</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">2,5µl</td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25, 26</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT32 TABLE [29862-30378] --> | ||
+ | <p> <strong>Sample preparation 29: Spore-amount test: GFP + strain 7 (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (0,5µl, 1,35g/ml) + strain 7 (amount: see table below, 400,000/µl) (90 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> centrifuged (2 min, 13400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> repeated steps 2 and 3)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (20µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubated on GOPTS-slide (2µl, 90 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and washed with PBS(1x) (10 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit33"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Amount (spores)</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2">25,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2">15,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2">10,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">25,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">15,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">10,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2">25,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2">15,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">WT(negative control)</td> | ||
+ | <td class="col2">10,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">25,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">15,000,000</td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25, 26</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">10,000,000</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT33 TABLE [30939-31526] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color8"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section10042016" id="section10042016">10/04/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (in EtOH resuspended)</strong> </p> | ||
+ | <p> –> spore concentration too high, too many layers of spores </p> | ||
+ | <p> <strong>Fluorescence microscopy 25 (Nikon C2+ confocal microscope): GFP + spores (in solution)</strong> </p> | ||
+ | <p> –> spore concentration too high, too many layers of spores </p> | ||
+ | <p> <strong>Fluorescence microscopy 26 (Nikon C2+ confocal microscope): Incubation test: GFP + spores (in solution)</strong> </p> | ||
+ | <p> –> spore concentration too high, too many layers of spores </p> | ||
+ | <p> Slide 1: </p> | ||
+ | <p> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/f/f5/T--Freiburg--targetingjournal18.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/a/a3/T--Freiburg--targetingjournal19.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/9/90/T--Freiburg--targetingjournal12.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/9/9f/T--Freiburg--targetingjournal21.png"> </center> | ||
+ | |||
+ | <br/> </p> | ||
+ | <p> 1.1) strain7 + GFP (30 min) (white field) 1.2) strain7 + GFP (30 min) (GFP-channel) 2.1) strain9 + GFP (30 min) (white field) 2.2) strain9 + GFP (30 min) (GFP-channel) | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> Slide 2: | ||
+ | <br/> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/6/61/T--Freiburg--targetingjournal22.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/be/T--Freiburg--targetingjournal23.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/88/T--Freiburg--targetingjournal24.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/1/13/T--Freiburg--targetingjournal25.png"> </center> | ||
+ | |||
+ | |||
+ | <br/> </p> | ||
+ | <p> 1.1) strain 7 + GFP (90 min)(white field) 1.2) strain 7 + GFP (90 min)(GFP-channel) 2.1) strain WT + GFP (90 min)(white field) 2.2) strain WT + GFP (90 min)(GFP-channel) | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> <strong>Fluorescence microscopy 27 (Nikon C2+ confocal microscope): GFP-volume test A: GFP + strain 7 (in solution)</strong> </p> | ||
+ | <p> –> background fluorescence was too high </p> | ||
+ | <p> <strong>Fluorescence microscopy 28 (Nikon C2+ confocal microscope): GFP-volume test B: GFP + strain 7 (in solution)</strong> </p> | ||
+ | <p> –> background fluorescence was too high </p> | ||
+ | <p> <strong>Fluorescence microscopy 29 (Nikon C2+ confocal microscope): GFP + strain 7 (in solution)</strong> </p> | ||
+ | <p> –> spores and WT show similar levels of fluorescence, probably autofluorescence </p> | ||
+ | <p> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/b/ba/T--Freiburg--targetingjournal26.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/83/T--Freiburg--targetingjournal27.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/8/88/T--Freiburg--targetingjournal28.png"> </center> | ||
+ | <center><img class="something" src="https://static.igem.org/mediawiki/2016/1/1b/T--Freiburg--targetingjournal29.png"> </center> | ||
+ | |||
+ | <br/> </p> | ||
+ | <p> 1) strain 7 + GFP (white field) 2) strain 7 + GFP (GFP-channel) 3) WT + GFP (white field) 4) WT + GFP (GFP-channel) | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> <strong>Sample preparation 30: Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (1µl, 1,35g/ml) + spores (strain 3,6,7,9) (25,000.000 spores) (90 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> step 2 and 3 repeated (2x/4x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in EtOH (250µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide (4µl, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit34"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Washing of GFP-spore solution</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">24</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">25</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">26</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">27</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT34 TABLE [34545-35086] --> | ||
+ | <p> <strong>Sample preparation 31: Wash-test B: GFP + spores (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with TBST(0,005%)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> steps 2 and 3 repeated (2x/4x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in EtOH (250µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide (4µl, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit35"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Washing of GFP-spore solution</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">2x</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 6</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + strain 9</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">GFP + WT (negative control)</td> | ||
+ | <td class="col2">4x</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">24</td> | ||
+ | <td class="col1">strain 3 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row14"> | ||
+ | <td class="col0">25</td> | ||
+ | <td class="col1">strain 6 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row15"> | ||
+ | <td class="col0">26</td> | ||
+ | <td class="col1">strain 7 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row16"> | ||
+ | <td class="col0">27</td> | ||
+ | <td class="col1">strain 9 (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT35 TABLE [35617-36158] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color1"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section10052016" id="section10052016">10/05/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 30 (Nikon C2+ confocal microscope): Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)</strong> </p> | ||
+ | <p> –> spore concentration too high, background fluorescence too high </p> | ||
+ | <p> <strong>Fluorescence microscopy 31 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (strains 3,6,7,9) (in solution)</strong> </p> | ||
+ | <p> –> indicates that strain 3 binds GFP </p> | ||
+ | <p> | ||
+ | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:1005.st3.gfp.slide27.waschtest4x.100ms60xj.jpg" class="media" title="labprotocol:1005.st3.gfp.slide27.waschtest4x.100ms60xj.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:1005.st3.gfp.slide27.waschtest4x.100ms60xj.jpg" class="medialeft" align="left" alt="" width="200" /></a> | ||
+ | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:1005.st3.gfp.slide27.waschtest4x.4s60xj.jpg" class="media" title="labprotocol:1005.st3.gfp.slide27.waschtest4x.4s60xj.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:1005.st3.gfp.slide27.waschtest4x.4s60xj.jpg" class="medialeft" align="left" alt="" width="200" /></a> | ||
+ | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:1005.wt.gfp.slide27.waschtest4x.100ms60xd.jpg" class="media" title="labprotocol:1005.wt.gfp.slide27.waschtest4x.100ms60xd.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:1005.wt.gfp.slide27.waschtest4x.100ms60xd.jpg" class="medialeft" align="left" alt="" width="200" /></a> | ||
+ | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:1005.wt.gfp.slide27.waschtest4x.4s60xd.jpg" class="media" title="labprotocol:1005.wt.gfp.slide27.waschtest4x.4s60xd.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:1005.wt.gfp.slide27.waschtest4x.4s60xd.jpg" class="medialeft" align="left" alt="" width="200" /></a> | ||
+ | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:1005.slide_27-wt_ohne_gfp-kontrolle.slide27-.4s60xi.jpg" class="media" title="labprotocol:1005.slide_27-wt_ohne_gfp-kontrolle.slide27-.4s60xi.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:1005.slide_27-wt_ohne_gfp-kontrolle.slide27-.4s60xi.jpg" class="medialeft" align="left" alt="" width="200" /></a> | ||
+ | <br/> </p> | ||
+ | <ul> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 1.1) GFP + strain 3 (white field)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 1.2) GFP + strain 3 (GFP-channel)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 2.1) GFP + WT (negative control) (white field)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 2.2) GFP + WT (negative control) (GFP-channel)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> 3) WT (negative control)</div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p> | ||
+ | <br/> </p> | ||
+ | </div> | ||
+ | <h3><a name="section10062016" id="section10062016">10/06/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 32: Wash-test C: GFP + spores (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with TBST(0,005%)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> steps 2 and 3 repeated (1x/3x/5x)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in EtOH (250µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide (4µl, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit36"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Washing of GFP-spore solution (step 3)</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">1x</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">1x</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">GFP + WT</td> | ||
+ | <td class="col2">1x</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">3x</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">3x</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">GFP + WT</td> | ||
+ | <td class="col2">3x</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP + strain 3</td> | ||
+ | <td class="col2">5x</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">GFP + strain 7</td> | ||
+ | <td class="col2">5x</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">GFP + WT</td> | ||
+ | <td class="col2">5x</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">WT (negative control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT36 TABLE [37646-37996] --> | ||
+ | </div> | ||
+ | <h3><a name="section10072016" id="section10072016">10/07/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 32 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (in solution)</strong> </p> | ||
+ | <p> –> spores and WT show similar levels of fluorescence, probably autofluorescence </p> | ||
+ | <p> <strong>Sample preparation 33: Blocking-test A: spores milk powder + GFP (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> spores washed twice with PBS(1x) (2x 100µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> spores resuspended and incubated in milk powder(5%) (100µl, incubation time: see table below)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with GFP (5µl, 1,35g/ml, 90 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with PBS(1x) (3x 100µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (20 µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation on GOPTS-slide (4µl, 30 min, 4°C)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed and blocked with milk powder(5%) (30 min, 10 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with PBS(1x) </div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> rinsed with ddH2O</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> dried with compressed air</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit37"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | <td class="col2"><strong>Incubation time</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">strain 3 + milk powder + GFP</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">strain 3 + milk powder + GFP</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">strain 7 + milk powder + GFP</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">strain 7 + milk powder + GFP</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">WT + milk powder + GFP (negative control)</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">WT + milk powder + GFP(negative control)</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">WT + milk powder (negative control)</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row8"> | ||
+ | <td class="col0">15, 16</td> | ||
+ | <td class="col1">WT + milk powder (negative control)</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row9"> | ||
+ | <td class="col0">17, 18</td> | ||
+ | <td class="col1">strain 3 + milk powder (negative control)</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row10"> | ||
+ | <td class="col0">19, 20</td> | ||
+ | <td class="col1">strain 3 + milk powder (negative control)</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row11"> | ||
+ | <td class="col0">21, 22</td> | ||
+ | <td class="col1">strain 7 + milk powder (negative control)</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | <tr class="row12"> | ||
+ | <td class="col0">23, 24</td> | ||
+ | <td class="col1">strain 7 + milk powder (negative control)</td> | ||
+ | <td class="col2">60 min</td> | ||
+ | </tr> | ||
+ | <tr class="row13"> | ||
+ | <td class="col0">25, 26</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | <td class="col2">30 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <!-- EDIT37 TABLE [38837-39543] --> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!-- para_20--> | ||
+ | </div> | ||
+ | <!-- color --> | ||
+ | <div class="color2"> | ||
+ | <div class="para_20"> | ||
+ | <h3><a name="section10082016" id="section10082016">10/08/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 33 (Nikon C2+ confocal microscope): Blocking-test A: spores + milk powder + GFP (in solution)</strong> </p> | ||
+ | <p> –> spores and WT show similar levels of fluorescence </p> | ||
+ | </div> | ||
+ | <h3><a name="section10112016" id="section10112016">10/11/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Sample preparation 34: Blocking-test B: spores + BSA + GFP (in solution)</strong> </p> | ||
+ | <ol> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> solution centrifuged (2 min, 13,400 rpm)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> spores (5,000,000) washed with PBS(1x) (3x 100µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> spores resuspended and incubated in BSA(5%) (100µl, 60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with GFP (50µl, 100µg/ml, 60 min)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> washed with TBST(0.05%) (5x 100µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> resuspended in PBS(1x) (50 µl)</div> | ||
+ | </li> | ||
+ | <li class="level1"> | ||
+ | <div class="li"> incubation with Mowiol DABCO (overnight)</div> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <div class="table sectionedit38"> | ||
+ | <table class="inline"> | ||
+ | <tr class="row0"> | ||
+ | <td class="col0"><strong>Spot No.</strong></td> | ||
+ | <td class="col1"><strong>Sample</strong></td> | ||
+ | </tr> | ||
+ | <tr class="row1"> | ||
+ | <td class="col0">1, 2</td> | ||
+ | <td class="col1">strain 3 + BSA + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row2"> | ||
+ | <td class="col0">3, 4</td> | ||
+ | <td class="col1">strain 7 + BSA + GFP</td> | ||
+ | </tr> | ||
+ | <tr class="row3"> | ||
+ | <td class="col0">5, 6</td> | ||
+ | <td class="col1">WT + BSA + GFP (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row4"> | ||
+ | <td class="col0">7, 8</td> | ||
+ | <td class="col1">WT + BSA (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row5"> | ||
+ | <td class="col0">9, 10</td> | ||
+ | <td class="col1">strain 3 + milk powder (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row6"> | ||
+ | <td class="col0">11, 12</td> | ||
+ | <td class="col1">strain 7 + milk powder (negative control)</td> | ||
+ | </tr> | ||
+ | <tr class="row7"> | ||
+ | <td class="col0">13, 14</td> | ||
+ | <td class="col1">GFP (positive control)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | <h3><a name="section10122016" id="section10122016">10/12/2016</a></h3> | ||
+ | <div class="level4"> | ||
+ | <p> <strong>Fluorescence microscopy 34 (Nikon C2+ confocal microscope): Blocking-test B: spores + BSA + GFP (in solution)</strong> </p> | ||
+ | <p> –> too little spores </p> | ||
+ | <div class="tags"><span> | ||
+ | <a href="/igem2016/doku.php?id=tag:lab_book" class="wikilink1" title="tag:lab_book" rel="tag">lab book</a> | ||
+ | </span></div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | <!-- | + | <!-- para_20--> |
</div> | </div> | ||
<!-- color --> | <!-- color --> |
Latest revision as of 23:20, 19 October 2016
Group 4 - Adhesion
07/04/2016
Bacillus subtilis strains used:
Strain |
B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5) |
B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5) |
B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5) |
B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5) |
B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5) |
07/05/2016
Slide preparation: GOPTS
Surface activation of glass slides by plasma generator, coated with GOPTS 3-glycidyloxypropyl trimethoxysilane, washed with acetone and then stored for up to two weeeks.
-
labeled with a diamond pen
-
washed with EtOH then ddH2O
-
dried slides with compressed air
-
activation of surface with plasma generator
-
incubation with GOPTS (overnight)
07/10/2016
Buffer for anti-GFP nanobody (10 ml)
-
61mg Tris
-
87mg NaCl
-
1ml Glycerin
-
2,5 ml Imidazole(1M)
-
fill up with ddH2O to 10 ml
07/12/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
-
incubation with nanobody (0.5µl, 330µg/ml, overnight, 4°C)
07/13/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
-
blocked with BSA(2%) (150µl/spot, 30 min)
-
washed with PBS(1x)
-
washed with imidazole (20mM) in PBS(1x)
-
washed with PBS(1x)
-
washed with ddH2O
-
dried with compressed air
-
incubation with B52-B56 (100µl/spot, 120 min)
-
incubation with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top
Spot No. | Sample |
1 | GOPTS + nanobody + B52 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + nanobody + B54 |
4 | GOPTS + nanobody + B55 |
5 | GOPTS + nanobody + B56 |
07/14/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
-
edges sealed with nail polish
Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56
–> pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
-
incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min)
-
blocked with BSA(2%) (90 min)
-
rinsed with PBS(1x)
-
stored in PBS(1x) (for 9 days)
Spot No. | Sample |
1 | GOPTS + nanobody + B52 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + nanobody + B54 |
4 | GOPTS + nanobody + B55 |
5 | GOPTS + nanobody + B56 |
6 | GOPTS + GFP (positive control) |
7 | GOPTS + WT (negative control) |
07/15/2016
Sample preparation
-
added nanobody to GOPTS plate (300 µg/ml, 5µl)
-
tried to pipette rest nanobody down, but didn´t work
-
blocked complete slide with BSA(2%)
07/23/016
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
-
washed slides with ddH2O
-
incubation with B52, B53, B54, B55, B56 (50µl/spot, overnight)
–> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP
07/24/2016
Preparation of samples 3: B53-B55
-
B53, B54, B55 (10µl/spot) on uncoated glass slide
-
Mowiol DABCO (5µl/spot)
-
covered with cover slip
Spot No. | Sample |
1 | B52 |
2 | B53 |
3 | B54 |
Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56
–> fluorescence signal of B53 and B54
Fluorescence microscopy 3 (Olympus 1×71): B53-B55
–> fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
-
incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d)
07/26/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
-
blocked with BSA(4%) (overnight)
07/27/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with water ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (positive control) |
2 | GOPTS + nanobody + GFP |
3 | GOPTS + BSA + GFP (negative control) |
4 | GOPTS + B53 (positive control) |
5 | GOPTS + B54 (positive control) |
6 | B53 (positive control) |
7 | B54 (positive control) |
07/28/2016
Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54
–> spores appear to not have been bound to nanobody
08/08/2016
Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP
-
incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min)
-
rinsed and blocked with BSA(4%) (30 min)
-
incubation with B53, B54, B55, B56, GFP (5µl/spot, 60 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with water ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl/spot, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + B55 (negative control) |
2 | GOPTS + B55 (negative control) |
3 | GOPTS + nanobody + B53 |
4 | GOPTS + nanobody + GFP (positive control) |
5 | GOPTS + BSA + GFP (negative control) |
6 | B53 (positive control) |
7 | B54 (positive control) |
8 | GOPTS + GFP (positive control) |
9 | GOPTS + B53 (positive control) |
10 | GOPTS + nanobody(Alexa647) + B53 |
11 | GOPTS + nanobody(Alexa647) + B54 |
12 | GOPTS + nanobody(Alexa647) + WT(negative control) |
13 | GOPTS + nanobody(Alexa647) + B56 (negative control) |
08/09/2016
Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP
–> spores have not been bound
08/10/2016
Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)
-
incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C)
-
centrifuged (13400 rpm, 2 min)
-
washed with PBS(1x) (1 ml)
-
repeated steps 2 and 3
-
centrifuged (13400 rpm, 2 min)
-
resuspended in PBS(1x) (400µl)
Spot No. | Sample |
1 | nanobody + WT (negative control) |
2 | nanobody + B53 |
3 | nanobody + B53 |
4 | nanobody + B55 (negative control) |
5 | nanobody + B56(negative control) |
6 | nanobody + GFP (positive control) |
7 | nanobody (positive control) |
8 | GFP (positive control) |
Fluorescence microscopy 6 (Nikon C2+ confocal microscope): anti-GFP nanobody + spores (WT, B53, B54, B56) in solution
–> no introduction to confocal mode: nanobody not observable
08/14/2016
Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54
-
incubated with nanobody (1µl, 330µg/ml, 60 min)
-
rinsed and blocked with BSA(4%) (60 min)
-
rinsed and washed twice with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with spores (10µl/spot, 120 min)
-
rinsed and washed twice with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | nanobody + WT (negative control) |
2 | nanobody + B53 |
3 | nanobody + B54 |
4 | nanobody + GFP (positive control) |
5 | nanobody (positive control) |
6 | GFP (positive control) |
Washing test A: GOPTS + anti-GFP nanobody + B53
-
incubation with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)
08/15/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
-
rinsed and blocked with BSA(2%) (60 min, 10 rpm)
-
rinsed with PBS(1x), washed PBS twice (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with B53 (10µl/spot, overnight)
08/16/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
-
rinsed and washed (see table below)
-
rinsed with ddh2O
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample | Condition |
1 | GOPTS + nanobody + B53 | washed 2x with PBS(1x) (10 min, 10 rpm) |
2 | GOPTS + nanobody + B53 | washed 3x with PBS(1x) (10 min, 10 rpm) |
3 | GOPTS + nanobody + B53 | washed 4x with PBS(1x) (10 min, 10 rpm) |
4 | GOPTS + nanobody + B53 | washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm) |
5 | GOPTS + nanobody + B53 | washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm) |
08/17/2016
Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54
–> spores have not bound to nanobody
Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53
–> spores have not bound to BSA
08/21/2016
Sample preparation 8: GOPTS + anti-GFP nanobody + GFP
-
incubation with nanobody (see table below, 60 min)
-
rinsed and blocked with BSA(4%) (60 min)
-
rinsed and washed with PBS(1x) (5 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with GFP (see table below, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (1µl) (positive control) |
2 | GOPTS + GFP (5 µl) (positive control) |
3 | GOPTS + nanobody (1µl, 330µg/ml) + GFP |
4 | GOPTS + nanobody (0.5µl, 10mg/ml) + GFP |
5, 6 | GOPTS + BSA + GFP (negative control) |
08/22/2016
Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP not bound, GOPTS plates too old
08/23/2016
Sample preparation 9: GOPTS + GFP
-
incubation with GFP (1.35mg/ml, volume/incubation: see table below)
-
rinsed and blocked with BSA(4%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (1 µl, 10 min) |
2 | GOPTS + GFP (2 µl, 10 min) |
3 | GOPTS + GFP (5 µl, 10 min) |
4 | GOPTS + GFP (1 µl, 15 min) |
5 | GOPTS + GFP (2 µl, 15 min) |
6 | GOPTS + GFP (5 µl, 15 min) |
7 | GOPTS + GFP (1 µl, 20 min) |
8 | GOPTS + GFP (2 µl, 20 min) |
9 | GOPTS + GFP (5 µl, 20 min) |
10 | GOPTS (negative control) |
Sample preparation 10: GOPTS + anti-GFP nanobody + B53
-
incubation with nanobody (1µl, 330µg/ml, 60 min)
-
rinsed and blocked with BSA(4%) (60 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with spores (10µl/spot, 30 min)
-
rinsed and washed with PBS(1x) (10min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + B53 |
2 | GOPTS + nanobody + B53 |
3 | GOPTS + GFP(positive control) |
4 | GOPTS + nanobody + GFP (positive control) |
5 | GOPTS + BSA + B53 (negative control) |
Washing test B: GOPTS + anti-GFP nanobody + B53
-
incubation with nanobody (1µl, 330µg/ml, 60 min)
-
rinsed and blocked with BSA(4%) (60 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with spores (10µl/spot, 30 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample | Condition |
1 | GOPTS + nanobody + B53 | rinsed with PBS(1x) |
2 | GOPTS + nanobody + B53 | swivel with PBS(1x) (0.5 min) |
3 | GOPTS + nanobody + B53 | washed with PBS(1x) (1 min, 10 rpm) |
4 | GOPTS + nanobody + B53 | washed with PBS(1x) (2 min, 10 rpm) |
5 | GOPTS + nanobody + B53 | washed with PBS(1x) (3 min, 10 rpm) |
6 | GOPTS + nanobody + B53 | washed with PBS(1x) (5 min, 10 rpm) |
7 | GOPTS + BSA + B53(negative control) | washed with PBS(1x) (10 min, 10 rpm) |
8 | GOPTS + BSA + GFP(negative control) | washed with PBS(1x) (10 min, 10 rpm) |
9 | GOPTS + GFP (positive control) | washed with PBS(1x) (10 min, 10 rpm) |
10 | GOPTS + B53 (positive control) | washed with PBS(1x) (10 min, 10 rpm) |
08/24/2016
Fluorescence microscopy 9 (Nikon C2+ confocal microscope): GOPTS + GFP
–> strong fluorescence signal at some of the edges: incubation too long/volume too little, GFP has dried and could not have been washed away properly
-
1) GOPTS + GFP (1 µl, 10 min)
-
2) GOPTS + GFP (2 µl, 10 min)
-
3) GOPTS + GFP (5 µl, 10 min)
-
4) GOPTS + GFP (1 µl, 15 min)
-
5) GOPTS + GFP (2 µl, 15 min)
-
6) GOPTS + GFP (5 µl, 15 min)
-
7) GOPTS + GFP (1 µl, 20 min)
-
8) GOPTS + GFP (2 µl, 20 min)
-
9) GOPTS + GFP (5 µl, 20 min)
Fluorescence microscopy 10 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53
–> no spores appear to have bound to nanobody
Fluorescence microscopy B (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53
–> no spores after washing, no binding between nanobody and B53?
08/25/2016
Blocking test C: GOPTS + BSA/milk powder + GFP
-
blocking with BSA(4%)/milk powder(5%) (120 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
incubation with GFP (1µl/spot, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + GFP (positive control) |
2 | GOPTS + milk powder(5%) + GFP |
3 | GOPTS + BSA(4%) + GFP |
Contamination Test D: BSA, PBS Mowiol DABCO
–> no contamination/pollution detected
Sample preparation 11: GOPTS + mCherry
-
incubated with mCherry (1µl, 2mg/ml, incubation: see table below)
-
rinsed and blocked with BSA(4%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + mCherry (5 min) |
2 | GOPTS + mCherry (10 min) |
3 | GOPTS + mCherry (15 min) |
4 | GOPTS (negative control) |
5 | mCherry + Mowiol DABCO (positive control) |
Sample preparation 12: GOPTS + anti-GFP nanobody + GFP
-
incubated with nanobody (5µl, 330µg/ml, 120 min)
-
rinsed and blocked with milk powder(5%) (60 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with GFP (2µl, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1 | GOPTS + nanobody + GFP |
2 | GOPTS (negative control) |
3 | GOPTS + GFP (positive control) |
4 | GOPTS + nanobody (negative control) |
5 | GOPTS + BSA + GFP (negative control) |
08/27/2016
Fluorescence microscopy C (Nikon C2+ confocal microscope): Blocking test: GOPTS + BSA/milkpowder + GFP
–> No GFP signal after blocking with BSA and milk powder
Fluorescence microscopy 11 (Nikon C2+ confocal microscope): GOPTS + mCherry
–> no mCherry-signal, too weak (quantum yield of mCherry about 1/3 of that of GFP)
Fluorescence microscopy 12 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP has not bound to nanobody
08/30/2016
Sample preparation 13: GOPTS + nanobody + GFP
-
incubated with nanobody (5µl, 330µg/ml, 60 min)
-
rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with GFP (2µl, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2 | GOPTS + nanobody(Alexa647) + GFP |
3,4 | GOPTS + nanobody + GFP |
5 | GOPTS + GFP (positive control) |
6 | GOPTS + nanobody (negative control) |
7 | GOPTS + BSA (negative control) |
09/01/2016
Fluorescence microscopy 13 (Nikon C2+ confocal microscope): GOPTS + nanobody + GFP
–> no GFP signal detectable
09/06/2016
Sample preparation 14: GOPTS + GFP
-
incubated with GFP (10µl, 1.35mg/ml, 30 min)
-
rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2 | GOPTS + mCherry |
3 | GOPTS + BSA + mCherry (negative control) |
4 | GOPTS + BSA (negative control) |
5 | mCherry (positive control) |
Mowiol was produced due to this protocol: https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf
09/07/2016
Fluorescence microscopy 14 (Nikon C2+ confocal microscope): GOPTS + GFP
–> spots with evenly distributed GFP
-
1) GOPTS + GFP
-
2) GOPTS + nanobody + GFP
09/14/2016
Sample preparation 16: GOPTS + B53
-
incubated with B53 (5µl, 60 min)
-
rinsed and blocked with milk powder(4%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1,2,3 | GOPTS + B53 |
4 | GOPTS + WT (negative control) |
5 | WT (negative control) |
6 | B53 (positive control) |
7 | GOPTS + GFP |
09/15/2016
Sample preparation 17: GOPTS + anti-GFP nanobody(Alexa647) + GFP
-
incubated with nanobody (5µl, 330µg/ml, 60 min)
-
rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with GFP (2µl, 1.35mg/ml, 20 min)
-
rinsed and washed with PBS(1x) (10 min, 10rpm)
-
rinsed with ddH20
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1-4 | GOPTS + nanobody(Alexa647) + GFP |
5 | GOPTS + milk powder + GFP (negative control) |
6 | GOPTS + milk powder + NB (negative control) |
7 | GOPTS + GFP |
8 | GOPTS (negative control) |
Fluorescence microscopy 16 (Nikon C2+ confocal microscope): GOPTS + B53
–> no spores detectable
09/16/2016
Fluorescence microscopy 17 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
–> GFP signal: GFP has been bound to nanobody
-
1-4) GOPTS + nanobody(Alexa647) + GFP
-
5) GOPTS + milk powder + GFP (negative control)
-
6) GOPTS + milk powder + NB (negative control)
-
7) GOPTS + GFP (positive control)
-
8) GOPTS (negative control)
09/17/2016
Sample preparation 18: GOPTS + anti-GFP nanobody + GFP (spotting-mask)
-
incubation with anti-GFP nanobody ( 2µl/spot, 330µg/ml, 10 min, 4°C)
-
rinsing and blocking with milk powder(5%) (15 min, 10 rpm)
-
rinsing with PBS(1x)
-
incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
(removed mask)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (overnight)
09/18/2016
Fluorescence microscopy 18 (Nikon C2+ confocal microscope): GOPTS + antiGFP-nanobody + GFP (spotting-mask)
–> no GFP signal detectable
Sample preparation 19: GOPTS + anti-GFP nanobody + GFP (spotting-mask)
-
incubation with anti-GFP nanobody (2µl/spot, 330µg/ml, 15 min, 4°C)
-
rinsing with PBS(1x)
-
(removed mask)
-
rinsing and blocking with milk powder(5%) (15 min, 2 rpm)
-
(reattached mask)
-
incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
-
(removed mask)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (overnight)
09/19/2016
Fluorescence microscopy 19 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP (spotting-mask)
–> slight GFP-signal on nanobody
Sample preparation 20: anti-GFP nanobody + B53 (in solution)
-
incubation of anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 15 min, 4°C) + B53
-
incubated on GOPTS-slide (2µl, 15 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)
–> problem: no washing away of unbound nanobody
Spot No. | Sample |
1,2 | B53 + nanobody(Alexa647) (incubated in solution) on GOPTS |
3,4 | WT + nanobody(Alexa647) (incubated in solution) on GOPTS (negative control) |
5,6 | B53 + nanobody(Alexa647) (incubated in solution) dried on milk powder |
7,8 | WT + nanobody(Alexa647) (incubated in solution) dried on milk powder (negative control) |
9,10 | GOPTS + nanobody(Alexa647) + GFP (positive control) |
11,12 | GOPTS + nanobody(Alexa647) + B53 |
13,14 | GOPTS + nanobody(Alexa647) + WT (negative control) |
15 | GOPTS + milk powder + nanobody(Alexa647) (negative control) |
16 | GOPTS + milk powder + B53 (negative control) |
17 | GOPTS + milk powder + WT (negative control) |
18 | GOPTS + GFP (positive control) |
19 | GOPTS + nanobody(Alexa647) (negative control) |
20 | B53 dried (positive control) |
09/20/2016
Sample preparation 21: anti-GFP nanobody + B53 (in solution)
-
incubation with anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 30 min, 4°C) + B53
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3
-
resuspended in PBS(1x) (30µl)
-
incubated on GOPTS-slide (2µl, 15 min)
-
rinsed and blocked with milkpowder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)
Spot No. | Sample |
1 | B53 + nanobody(Alexa647) (incubated in solution) on GOPTS |
2 | WT + nanobody(Alexa647) (incubated in solution) on GOPTS |
3,4 | B53 + nanobody(Alexa647) dried on milkpowder |
5,6 | WT + nanobody(Alexa647) dried on milkpowder |
09/29/2016
Sample preparation 22: GOPTS + GFP
-
incubation with GFP (1 µl, 1.35mg/ml, 30 min)
-
rinsed and blocked with milk powder(5%) (30 min)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (5µl, overnight)
Spot No. | Sample |
1-5 | GOPTS + GFP |
6 | GOPTS |
09/30/2016
–> Construct spores are ready for analysis
Strain No. | Strain |
3 | 150: aGFPnano_HA_aHelix_cgeA |
6 | 151: aGFPnano_HA_aHelix_cotG |
7 | 159: aGFPnano_HA_G4S_CotZ |
9 | 157: aGFPnano_HA_aHelix_CotZ |
Fluorescence microscopy 22 (Nikon C2+ confocal microscope): GOPTS + GFP
–> spots with evenly distributed GFP
-
incubation with GFP (1 µl, 1.35mg/ml, 30 min)
-
spores centrifuged (2 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 1 repeated
-
resuspended in PBS(1x)
-
incubation on GOPTS-slide(GFP) with spores (4µl, 400,000/µl, incubation time: see table below, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Incubation time (min) |
1 | GOPTS + GFP + strain 3 | 5 |
2 | GOPTS + GFP + strain 3 | 10 |
3 | GOPTS + GFP + strain 3 | 15 |
4 | GOPTS + GFP + strain 6 | 5 |
5 | GOPTS + GFP + strain 6 | 10 |
6 | GOPTS + GFP + strain 6 | 15 |
7 | GOPTS + GFP + strain 7 | 5 |
8 | GOPTS + GFP + strain 7 | 10 |
9 | GOPTS + GFP + strain 7 | 15 |
10 | GOPTS + GFP + strain 9 | 5 |
11 | GOPTS + GFP + strain 9 | 10 |
12 | GOPTS + GFP + strain 9 | 15 |
13, 14 | GOPTS + GFP (positive control) | 10 |
15 | GOPTS + GFP + WT (negative control) | 5 |
16 | GOPTS + GFP + WT (negative control) | 10 |
17 | GOPTS + GFP + WT (negative control) | 15 |
18 | GOPTS + strain 3 (positive control) | 10 |
19 | GOPTS + strain 6 (positive control) | 10 |
20 | GOPTS + strain 7 (positive control) | 10 |
21 | GOPTS + strain 9 (positive control) | 10 |
22 | GOPTS + WT (negative control) | 10 |
10/01/2016
Sample preparation 23: GOPTS + GFP + spores
–> no spores detectable
Sample preparation 24: GOPTS + GFP + spores (in EtOH resuspended)
-
incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (10 min, 4°C)
-
solution centrifuged (4 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 2 repeated
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample |
1-3 | GOPTS + GFP + strain 6 |
4-6 | GOPTS + GFP + WT |
7-9 | GOPTS + GFP |
10-12 | GOPTS + strain 6 |
13-15 | GOPTS + WT |
10/02/2016
Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (strains 3,6,7,9,wt) (in EtOH resuspended)
–> EtOH denaturated GFP: very weak signal. No spores detectable
Sample preparation 25: GFP + spores (strains 3,6,7,9) (in solution)
-
incubation of GFP (4µl, 1,35g/ml) + spores(strain 3,6,7,9) (4µl, 400,000/µl) (incubation time: see table below, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (4µl, 10 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubrnight)ation with Mowiol DABCO
Spot No. | Sample |
1, 2 | GFP + strain 3 |
3, 4 | GFP + strain 6 |
5, 6 | GFP + strain 7 |
7, 8 | GFP + strain 9 |
9, 10 | GFP + WT (negative control) |
11, 12 | GFP + PBS (positive control) |
13, 14 | GFP (positive control) |
15, 16 | WT (negative control) |
17 | strain 3 (negative control) |
18 | strain 6 (negative control) |
19 | strain 7 (negative control) |
20 | strain 9 (negative control) |
10/03/2016
Sample preparation 26: Incubation test: GFP + spores (in solution)
-
incubation of GFP (4µl, 1,35g/ml) + spores (4µl, 400,000/µl) (incubation time: see tables below, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl))
-
incubated on GOPTS-slide (4µl, 10 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Slide 1:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 30 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 30 min |
5, 6 | GFP + strain 6 | 30 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 30 min |
9, 10 | GFP + strain 7 | 30 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 30 min |
13, 14 | GFP + strain 9 | 30 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 30 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Slide 2:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 60 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 60 min |
5, 6 | GFP + strain 6 | 60 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 60 min |
9, 10 | GFP + strain 7 | 60 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 60 min |
13, 14 | GFP + strain 9 | 60 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 60 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Slide 3:
Spot No. | Sample | Incubation time |
1, 2 | GFP + strain 3 | 120 min |
3, 4 | GFP + strain 3(pellet washed with PBS(1x)) | 120 min |
5, 6 | GFP + strain 6 | 120 min |
7, 8 | GFP + strain 6(pellet washed with PBS(1x)) | 120 min |
9, 10 | GFP + strain 7 | 120 min |
11, 12 | GFP + strain 7(pellet washed with PBS(1x)) | 120 min |
13, 14 | GFP + strain 9 | 120 min |
15, 16 | GFP + strain 9 (pellet washed withn PBS(1x)) | 120 min |
17, 18 | GFP + WT (negative control) | |
19, 20 | GFP + WT (negative control) (pellet washed with PBS(1x)) | |
21, 22 | GFP (positive control) | |
23, 24 | WT (negative control) | |
25 | strain 3 (negative control) | |
26 | strain 6 (negative control) | |
27 | strain 7 (negative control) | |
28 | strain 9 (negative control) |
Sample preparation 28: GFP-volume test B: GFP + strain 7 (in solution, washed)
-
incubation of GFP (volume: see table below, 1,35g/ml) + strain 7 (4µl, 400,000/µl) (90 min, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (2µl, 90 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Volume (GFP) |
1, 2 | GFP + strain 7 | 0,5µl |
3, 4 | GFP + strain 7 | 1,0µl |
5, 6 | GFP + strain 7 | 1,5µl |
7, 8 | GFP + strain 7 | 2,0µl |
9, 10 | GFP + strain 7 | 2,5µl |
11, 12 | GFP (positive control) | 4,0µl |
13, 14 | strain 7 (negative control) | |
15, 16 | GFP + WT (negative control) | 0,5µl |
17, 18 | GFP + WT (negative control) | 1,0µl |
19, 20 | GFP + WT (negative control) | 1,5µl |
21, 22 | GFP + WT (negative control) | 2,0µl |
23, 24 | GFP + WT (negative control) | 2,5µl |
25, 26 | WT (negative control) |
Sample preparation 29: Spore-amount test: GFP + strain 7 (in solution)
-
incubation of GFP (0,5µl, 1,35g/ml) + strain 7 (amount: see table below, 400,000/µl) (90 min, 4°C)
-
centrifuged (2 min, 13400 rpm)
-
washed with PBS(1x)
-
repeated steps 2 and 3)
-
resuspended in PBS(1x) (20µl)
-
incubated on GOPTS-slide (2µl, 90 min)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Amount (spores) |
1, 2 | strain 7 (negative control) | 25,000,000 |
3, 4 | strain 7 (negative control) | 15,000,000 |
5, 6 | strain 7 (negative control) | 10,000,000 |
7, 8 | GFP + strain 7 | 25,000,000 |
9, 10 | GFP + strain 7 | 15,000,000 |
11, 12 | GFP + strain 7 | 10,000,000 |
13, 14 | GFP (positive control) | |
15, 16 | WT (negative control) | 25,000,000 |
17, 18 | WT (negative control) | 15,000,000 |
19, 20 | WT(negative control) | 10,000,000 |
21, 22 | GFP + WT (negative control) | 25,000,000 |
23, 24 | GFP + WT (negative control) | 15,000,000 |
25, 26 | GFP + WT (negative control) | 10,000,000 |
10/04/2016
Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (in EtOH resuspended)
–> spore concentration too high, too many layers of spores
Fluorescence microscopy 25 (Nikon C2+ confocal microscope): GFP + spores (in solution)
–> spore concentration too high, too many layers of spores
Fluorescence microscopy 26 (Nikon C2+ confocal microscope): Incubation test: GFP + spores (in solution)
–> spore concentration too high, too many layers of spores
Slide 1:
1.1) strain7 + GFP (30 min) (white field) 1.2) strain7 + GFP (30 min) (GFP-channel) 2.1) strain9 + GFP (30 min) (white field) 2.2) strain9 + GFP (30 min) (GFP-channel)
Slide 2:
1.1) strain 7 + GFP (90 min)(white field) 1.2) strain 7 + GFP (90 min)(GFP-channel) 2.1) strain WT + GFP (90 min)(white field) 2.2) strain WT + GFP (90 min)(GFP-channel)
Fluorescence microscopy 27 (Nikon C2+ confocal microscope): GFP-volume test A: GFP + strain 7 (in solution)
–> background fluorescence was too high
Fluorescence microscopy 28 (Nikon C2+ confocal microscope): GFP-volume test B: GFP + strain 7 (in solution)
–> background fluorescence was too high
Fluorescence microscopy 29 (Nikon C2+ confocal microscope): GFP + strain 7 (in solution)
–> spores and WT show similar levels of fluorescence, probably autofluorescence
1) strain 7 + GFP (white field) 2) strain 7 + GFP (GFP-channel) 3) WT + GFP (white field) 4) WT + GFP (GFP-channel)
Sample preparation 30: Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (strain 3,6,7,9) (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with PBS(1x)
-
step 2 and 3 repeated (2x/4x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution |
1, 2 | GFP + strain 3 | 2x |
3, 4 | GFP + strain 6 | 2x |
5, 6 | GFP + strain 7 | 2x |
7, 8 | GFP + strain 9 | 2x |
9, 10 | GFP + WT (negative control) | 2x |
11, 12 | GFP + strain 3 | 4x |
13, 14 | GFP + strain 6 | 4x |
15, 16 | GFP + strain 7 | 4x |
17, 18 | GFP + strain 9 | 4x |
19, 20 | GFP + WT (negative control) | 4x |
21, 22 | GFP (positive control) | |
23 | WT (negative control) | |
24 | strain 3 (negative control) | |
25 | strain 6 (negative control) | |
26 | strain 7 (negative control) | |
27 | strain 9 (negative control) |
Sample preparation 31: Wash-test B: GFP + spores (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with TBST(0,005%)
-
steps 2 and 3 repeated (2x/4x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution |
1, 2 | GFP + strain 3 | 2x |
3, 4 | GFP + strain 6 | 2x |
5, 6 | GFP + strain 7 | 2x |
7, 8 | GFP + strain 9 | 2x |
9, 10 | GFP + WT (negative control) | 2x |
11, 12 | GFP + strain 3 | 4x |
13, 14 | GFP + strain 6 | 4x |
15, 16 | GFP + strain 7 | 4x |
17, 18 | GFP + strain 9 | 4x |
19, 20 | GFP + WT (negative control) | 4x |
21, 22 | GFP (positive control) | |
23 | WT (negative control) | |
24 | strain 3 (negative control) | |
25 | strain 6 (negative control) | |
26 | strain 7 (negative control) | |
27 | strain 9 (negative control) |
10/05/2016
Fluorescence microscopy 30 (Nikon C2+ confocal microscope): Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)
–> spore concentration too high, background fluorescence too high
Fluorescence microscopy 31 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (strains 3,6,7,9) (in solution)
–> indicates that strain 3 binds GFP
-
1.1) GFP + strain 3 (white field)
-
1.2) GFP + strain 3 (GFP-channel)
-
2.1) GFP + WT (negative control) (white field)
-
2.2) GFP + WT (negative control) (GFP-channel)
-
3) WT (negative control)
10/06/2016
Sample preparation 32: Wash-test C: GFP + spores (in solution)
-
incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
-
solution centrifuged (2 min, 13,400 rpm)
-
washed with TBST(0,005%)
-
steps 2 and 3 repeated (1x/3x/5x)
-
resuspended in EtOH (250µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Washing of GFP-spore solution (step 3) |
1, 2 | GFP + strain 3 | 1x |
3, 4 | GFP + strain 7 | 1x |
5, 6 | GFP + WT | 1x |
7, 8 | GFP + strain 3 | 3x |
9, 10 | GFP + strain 7 | 3x |
11, 12 | GFP + WT | 3x |
13, 14 | GFP + strain 3 | 5x |
15, 16 | GFP + strain 7 | 5x |
17, 18 | GFP + WT | 5x |
19, 20 | WT (negative control) | |
21, 22 | GFP (positive control) |
10/07/2016
Fluorescence microscopy 32 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (in solution)
–> spores and WT show similar levels of fluorescence, probably autofluorescence
Sample preparation 33: Blocking-test A: spores milk powder + GFP (in solution)
-
solution centrifuged (2 min, 13,400 rpm)
-
spores washed twice with PBS(1x) (2x 100µl)
-
spores resuspended and incubated in milk powder(5%) (100µl, incubation time: see table below)
-
incubation with GFP (5µl, 1,35g/ml, 90 min)
-
washed with PBS(1x) (3x 100µl)
-
resuspended in PBS(1x) (20 µl)
-
incubation on GOPTS-slide (4µl, 30 min, 4°C)
-
rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
-
rinsed with PBS(1x)
-
rinsed with ddH2O
-
dried with compressed air
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample | Incubation time |
1, 2 | strain 3 + milk powder + GFP | 30 min |
3, 4 | strain 3 + milk powder + GFP | 60 min |
5, 6 | strain 7 + milk powder + GFP | 30 min |
7, 8 | strain 7 + milk powder + GFP | 60 min |
9, 10 | WT + milk powder + GFP (negative control) | 30 min |
11, 12 | WT + milk powder + GFP(negative control) | 60 min |
13, 14 | WT + milk powder (negative control) | 30 min |
15, 16 | WT + milk powder (negative control) | 60 min |
17, 18 | strain 3 + milk powder (negative control) | 30 min |
19, 20 | strain 3 + milk powder (negative control) | 60 min |
21, 22 | strain 7 + milk powder (negative control) | 30 min |
23, 24 | strain 7 + milk powder (negative control) | 60 min |
25, 26 | GFP (positive control) | 30 min |
10/08/2016
Fluorescence microscopy 33 (Nikon C2+ confocal microscope): Blocking-test A: spores + milk powder + GFP (in solution)
–> spores and WT show similar levels of fluorescence
10/11/2016
Sample preparation 34: Blocking-test B: spores + BSA + GFP (in solution)
-
solution centrifuged (2 min, 13,400 rpm)
-
spores (5,000,000) washed with PBS(1x) (3x 100µl)
-
spores resuspended and incubated in BSA(5%) (100µl, 60 min)
-
incubation with GFP (50µl, 100µg/ml, 60 min)
-
washed with TBST(0.05%) (5x 100µl)
-
resuspended in PBS(1x) (50 µl)
-
incubation with Mowiol DABCO (overnight)
Spot No. | Sample |
1, 2 | strain 3 + BSA + GFP |
3, 4 | strain 7 + BSA + GFP |
5, 6 | WT + BSA + GFP (negative control) |
7, 8 | WT + BSA (negative control) |
9, 10 | strain 3 + milk powder (negative control) |
11, 12 | strain 7 + milk powder (negative control) |
13, 14 | GFP (positive control) |
10/12/2016
Fluorescence microscopy 34 (Nikon C2+ confocal microscope): Blocking-test B: spores + BSA + GFP (in solution)
–> too little spores