Difference between revisions of "Team:Freiburg/NotebookTargeting"
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| − | </ | + | <head> |
| + | <style> | ||
| + | .level3 { | ||
| + | font-size: 18px; | ||
| + | } | ||
| + | |||
| + | .pinkpurple { | ||
| + | background-color: #871150; | ||
| + | color: orange; | ||
| + | } | ||
| + | |||
| + | h3 { | ||
| + | font-size: 36px; | ||
| + | padding: 20px; | ||
| + | } | ||
| + | |||
| + | strong { | ||
| + | color: grey; | ||
| + | } | ||
| + | |||
| + | .grey { | ||
| + | background-color: grey; | ||
| + | color: orange; | ||
| + | } | ||
| + | |||
| + | .para_20 { | ||
| + | padding: 5% 15%; | ||
| + | } | ||
| + | |||
| + | .image img { | ||
| + | margin: 20px; | ||
| + | width: 180px; | ||
| + | } | ||
| + | |||
| + | .image img:hover { | ||
| + | cursor: pointer; | ||
| + | -webkit-transform: scale(1.1); | ||
| + | /* Safari and Chrome */ | ||
| + | -moz-transform: scale(1.1); | ||
| + | /* Firefox */ | ||
| + | transition-duration: .2s; | ||
| + | transition-timing-function: linear; | ||
| + | } | ||
| + | </style> | ||
| + | </head> | ||
| − | </div> | + | <body> |
| + | <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" style="width:200px"> </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" style="width:200px"> </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" style="width:200px"> </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" style="width:200px"> </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" style="width:200px"> </a> | ||
| + | </div> | ||
| + | </center> | ||
| + | </div> | ||
| + | <div class="color8"> | ||
| + | <div class="para_center_20"> | ||
| + | <div class="level1"> | ||
| + | <h1 class="sectionedit1"><a name="group_4" id="group_4">Group 4</a></h1> | ||
| + | <p> <strong>Members:Kevin, Christian, Vivi, Nathalie</strong> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="color2"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section07042016" id="section07042016">07/04/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Preparation of overnight cultures of 5 different <em>Bacillus subtilis</em> strains with GFP on CotZ-fusionprotein</strong> </p> | ||
| + | <div class="table sectionedit2"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Nr.</td> | ||
| + | <td class="col1">Strain</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5)</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5)</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT2 TABLE [206-520] --> | ||
| + | <p> <strong>Medium:</strong> Preparation of LB-medium with <em>Chloramphenicol</em>: 50µl(1000X) in 50ml LB-Medium | ||
| + | <br/> </p> | ||
| + | <p> <strong>Inoculation:</strong> 5 reaction tubes with 5 ml LB-<em>Chloramphenicol</em>-medium have been used. Each has been inoculated with a little piece of filter paper with spores. Cultures have been incubated at 200rpm and 37°C, 16:10Pm to 8am. No growth has been observed ! </p> | ||
| + | <p> Further spore production was performed by group 2. </p> | ||
| + | </div> | ||
| + | <h3><a name="section07052016" id="section07052016">07/05/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Preparation of GOPTS coated glass slides</strong> </p> | ||
| + | <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> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> labeled with a diamond pen</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with EtOH then ddH2O</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> dried slides with compressed air</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> activation of surface with plasma generator</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with GOPTS (overnight)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section07102016" id="section07102016">07/10/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Buffer for anti-GFP-nanobody (10 ml)</strong> </p> | ||
| + | <ul> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 61mg Tris </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 87mg NaCl </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 1ml Glycerin </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 2,5 ml Imidazole(1M)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> fill up with ddH2O to 10 ml</div> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color3"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section07122016" id="section07122016">07/12/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Glass slides were spotted with 0,5 µl nanobody (300µg/ml) (Spot location marked with permanent marker.)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Slides were incubated over night in the fridge at 4°C in darkness</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 150µl BSA(2x) on each spot, 30 min incubation</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with 20mM imidazole in PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with ddH2o</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> dried with compressed air </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 100µl of spore solution on each slide</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section07132016" id="section07132016">07/13/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> blocked with BSA(2%) (150µl/spot, 30 min) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with imidazole (20mM) in PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with ddH2O</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> dried with compressed air </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with B52-B56 (100µl/spot, 120 min) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit3"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B52</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B54</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B55</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B56</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT3 TABLE [2479-2639] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | <h3><a name="section07142016" id="section07142016">07/14/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56</strong> | ||
| + | <br/> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> edges sealed with nail polish</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> <strong>Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56</strong> </p> | ||
| + | <p> –> pictures inconclusive:pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls | ||
| + | <br/> </p> | ||
| + | <p> <strong>Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56</strong> | ||
| + | <br/> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> blocked with BSA(2%) (90 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed with PBS(1x)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> stored in PBS(1x) (for 9 days)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit4"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B52</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B54</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B55</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B56</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">GOPTS + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">GOPTS + WT (negative control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT4 TABLE [3198-3429] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color4"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section07152016" id="section07152016">07/15/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> added nanobody to GOPTS plate (300 µg/ml, 5µl)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> tried to pipette rest nanobody down, but didn´t work</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> blocked complete slide with BSA(2%)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section0723016" id="section0723016">07/23/016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> | ||
| + | <br/> <strong>Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Washed slides with ddH2O</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Add spores (50µl/spot)(B52,B53,B54,B55,B65)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> | ||
| + | <br/> –> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP </p> | ||
| + | <p> <strong>Coating of new slides with GOPTS (experiment 3)</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> according to protocol</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section07242016" id="section07242016">07/24/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Preparation of samples 3: B53-B55</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> B53, B54, B55 (10µl/spot) on uncoated glass slide</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Mowiol DABCO (5µl/spot)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> covered with cover slip</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit5"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B52</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B54</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT5 TABLE [4121-4225] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <p> <strong>Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> fluorescence signal of B53 and B54 </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> <strong>Fluorescence microscopy 3 (Olympus 1×71): B53-B55</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> B53 sample: no fluorescence observable (Air bubble) | ||
| + | <br/> B54 sample: no fluorescence observable | ||
| + | <br/> </p> | ||
| + | <p> <strong>Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d) </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color5"> | ||
| + | <div class="para_20"> | ||
| + | <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> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Prepared new BSA solution with higher concentration (4% w/v)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Blocked slides with BSA (4%) ,covered complete slide with BSA</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> inkubated over night</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section07272016" id="section07272016">07/27/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
| + | <ol> | ||
| + | <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 water 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/spot, overnight)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> *Positive controls: </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Slide with spores B53–> fuorescence observable</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> B54 –>Fluorescence observable </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit6"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</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">2 GOPTS + nanobody + GFP</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + B53 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + B54 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">B53 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">B54 (positive control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT6 TABLE [5340-5596] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <p> <strong>Slides of experiment 3 </strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Washing with PBS</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed with water ddH2O</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> dried with compressed air</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> <strong>Positive controls exp. 3:</strong> </p> | ||
| + | <ul> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> GFP direct on GOPTS</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> GFP on nanobody</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> GFP on BSA blocked surface</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Spores direct on GOPTS</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> double concentrated spores direct on glass slide B53 and B54</div> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <p> Spores were added to glass surface and incubated until its dry </p> | ||
| + | </div> | ||
| + | <h3><a name="section07282016" id="section07282016">07/28/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <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> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Washed slides of exp 3 with PBS</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> added DABCO (5µl)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color6"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section07292016" id="section07292016">07/29/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>prepared samples for fluorescence microscopy</strong> </p> | ||
| + | </div> | ||
| + | <h3><a name="section08082016" id="section08082016">08/08/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(4%) (30 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation with B53, B54, B55, B56, GFP (5µl/spot, 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 water 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/spot, overnight)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit7"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B55 (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + B55 (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">B53 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">B54 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row8"> | ||
| + | <td class="col0">8.</td> | ||
| + | <td class="col1">GOPTS + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row9"> | ||
| + | <td class="col0">9.</td> | ||
| + | <td class="col1">GOPTS + B53 (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row10"> | ||
| + | <td class="col0">10.</td> | ||
| + | <td class="col1">GOPTS + nanobody(Alexa647) + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row11"> | ||
| + | <td class="col0">11.</td> | ||
| + | <td class="col1">GOPTS + nanobody(Alexa647) + B54</td> | ||
| + | </tr> | ||
| + | <tr class="row12"> | ||
| + | <td class="col0">12.</td> | ||
| + | <td class="col1">GOPTS + nanobody(Alexa647) + WT(negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row13"> | ||
| + | <td class="col0">13.</td> | ||
| + | <td class="col1">GOPTS + nanobody(Alexa647) + B56 (negative control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT7 TABLE [6696-7236] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | <h3><a name="section08092016" id="section08092016">08/09/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Test if spores bind Nanobody in solution</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Wt, B53, B54, B56 centrifuged, resuspended in 50 µl nanobody-buffer, </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> add 1µl labeled nanobody </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated 1h at 4°C</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed 2 times 10 min with PBS</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> <strong>Problem:</strong> no introduction in the confocal microscopy mode </p> | ||
| + | <ul> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> Flourescence microscopy </div> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <p> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:glass_b53_alexananobody_2016.08.09.jpg" class="media" title="labprotocol:glass_b53_alexananobody_2016.08.09.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:glass_b53_alexananobody_2016.08.09.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:gopts_nb_b55_wl.jpg" class="media" title="labprotocol:gopts_nb_b55_wl.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:gopts_nb_b55_wl.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:gopts_nb_b53_wl.jpg" class="media" title="labprotocol:gopts_nb_b53_wl.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:gopts_nb_b53_wl.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:gopts_gfp.jpg" class="media" title="labprotocol:gopts_gfp.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:gopts_gfp.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | </p> | ||
| + | <p> <strong>Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP</strong> </p> | ||
| + | <p> –>only fluorescence detectable on slides 6 and 8 </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color7"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section08102016" id="section08102016">08/10/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> centrifuged (13400 rpm, 2 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> washed with PBS(1x) (1 ml)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> repeated steps 2 and 3</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> centrifuged (13400 rpm, 2 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> resuspended in PBS(1x) (400µl)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit8"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">nanobody + WT (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">nanobody + B55 (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">nanobody + B56(negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">nanobody + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">nanobody (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row8"> | ||
| + | <td class="col0">8.</td> | ||
| + | <td class="col1">GFP (positive control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT8 TABLE [8315-8590] --> | ||
| + | <p> | ||
| + | <br/> </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> <strong>Light microscopy</strong> Analyse the slides from 28.7.2016 under light mikroscope </p> | ||
| + | </div> | ||
| + | <h3><a name="section08142016" id="section08142016">08/14/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with nanobody (1µl, 330µg/ml, 60 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(4%) (60 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and washed twice 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 spores (10µl/spot, 120 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and washed twice 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 sectionedit9"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">nanobody + WT (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">nanobody + B54</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">nanobody + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">nanobody (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">GFP (positive control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT9 TABLE [9362-9560] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <p> <strong>Washing test:</strong> We want to test if spores (B53) really bind to BSA surface like the previous test showed. We also want to test if the washing procedure has an impact on this result. </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> 5 slides coated with Gopts</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> added 1 µl Nanobody (333µg/ml)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated over night</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> | ||
| + | <br/> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <h3><a name="section08152016" id="section08152016">08/15/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> | ||
| + | <br/> </p> | ||
| + | <p> <strong>Hier Bild von BSA + B53 mit GFP-signal</strong> | ||
| + | <br/> | ||
| + | <br/> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(2%) (60 min, 10 rpm) </div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed with PBS(1x), washed PBS twice (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 B53 (10µl/spot, overnight)</div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color8"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section08162016" id="section08162016">08/16/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Washing test A: GOPTS + anti-GFP nanobody + B53</strong> </p> | ||
| + | <p> <strong>conditions</strong> | ||
| + | <br/> Coated plates were rinsed with PBS to remove the spore soluten. Slides were put in PBS in Petri dishes and put on the shaker. | ||
| + | <br/> </p> | ||
| + | <div class="table sectionedit10"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | <td class="col2">Condition</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed 2x with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed 3x with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed 4x with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed 1x with PBS(1x) + 0,05% Tween20 (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed 1x with PBS(1x) + 0,1 % Tween20 (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT10 TABLE [10530-10932] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | <h3><a name="section17082016" id="section17082016">17/08/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54</strong> </p> | ||
| + | <p> –> spores have not bound to nanobody | ||
| + | <br/> </p> | ||
| + | <p> <strong>Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53</strong> –>spores have not bound to BSA, picture of BSA + B53 must have been a mistake | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | <h3><a name="section08212016" id="section08212016">08/21/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 8: GOPTS + anti-GFP nanobody + GFP</strong> </p> | ||
| + | <p> –> 10 slides activated with plasmagenerator coated with GOPTS. </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with nanobody (see table below, 60 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(4%) (60 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and washed with PBS(1x) (5 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 (see table below, 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) | ||
| + | <br/> </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <div class="table sectionedit11"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + GFP (1µl) (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + GFP (5 µl) (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody (1µl, 330µg/ml) + GFP</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody (0.5µl, 10mg/ml) + GFP</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">GOPTS + BSA + GFP (negative control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT11 TABLE [11856-12139] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <p> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_1.5_2_10s_0_5ulnbunverduennt_.jpg" class="media" title="labprotocol:freigem_1.5_2_10s_0_5ulnbunverduennt_.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_1.5_2_10s_0_5ulnbunverduennt_.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_2016_1.1_21.08.1ulgfpauf_gpots.jpg" class="media" title="labprotocol:freigem_2016_1.1_21.08.1ulgfpauf_gpots.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_2016_1.1_21.08.1ulgfpauf_gpots.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_1.5_3_20s_0_5ulnbunverduennt_plusgfp.jpg" class="media" title="labprotocol:freigem_1.5_3_20s_0_5ulnbunverduennt_plusgfp.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_1.5_3_20s_0_5ulnbunverduennt_plusgfp.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_2016_1.2_10s_1ulnb1.30verdplus_gfp.jpg" class="media" title="labprotocol:freigem_2016_1.2_10s_1ulnb1.30verdplus_gfp.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_2016_1.2_10s_1ulnb1.30verdplus_gfp.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_2016_1.4_10s5ulgfpaufgopts.jpg" class="media" title="labprotocol:freigem_2016_1.4_10s5ulgfpaufgopts.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_2016_1.4_10s5ulgfpaufgopts.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:freigem_2016_1.5_10s_0_5ulnbunverduennt.jpg" class="media" title="labprotocol:freigem_2016_1.5_10s_0_5ulnbunverduennt.jpg"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:freigem_2016_1.5_10s_0_5ulnbunverduennt.jpg" class="mediacenter" alt="" width="200" /></a> | ||
| + | </p> | ||
| + | <p> Picture after editing of shadow and contrast: | ||
| + | <a href="/igem2016/lib/exe/detail.php?id=labprotocol%3Amagic_bullets&media=labprotocol:gfp2.png" class="media" title="labprotocol:gfp2.png"><img src="/igem2016/lib/exe/fetch.php?w=200&media=labprotocol:gfp2.png" class="mediacenter" alt="" width="200" /></a> Bedingungen: 5µl,GFP,20min. incubation, mit BSA blocking, 5min. waschen mit PBS, </p> | ||
| + | <p> <strong>Blocking Test</strong> We want to test if the GOPTS is properly blocked by BSA and if milkprotein is also usefull for blocking </p> | ||
| + | <p> 1 Slide with : </p> | ||
| + | <div class="table sectionedit12"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">GFP auf GOPTS</td> | ||
| + | <td class="col1" colspan="2">GFP good visible , border clearly visible</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">Milkprotein on GOPTS+GFP</td> | ||
| + | <td class="col1" colspan="2">No GFP observable</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">BSA on GOPTS + GFP</td> | ||
| + | <td class="col1" colspan="2">NO GFP observalbe</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT12 TABLE [12855-12999] --> | ||
| + | <p> <strong>Contamination Test</strong> To avoid wrong interpretations of the fluorescence pictures we tested the chemikals and solutions we use, to find out if there are fluorescent contaminations. </p> | ||
| + | <div class="table sectionedit13"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">solution</td> | ||
| + | <td class="col1">result (GFP LASER)</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">Mowiol/DABCO</td> | ||
| + | <td class="col1">a vew fluorecent crystals</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">PBS</td> | ||
| + | <td class="col1">a vew fluorecent crystals</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">BSA</td> | ||
| + | <td class="col1">no fluorescence</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT13 TABLE [13185-13309] --> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | <!-- para20 --> | ||
| + | </div> | ||
| + | <!-- color --> | ||
| + | <div class="color1"> | ||
| + | <div class="para_20"> | ||
| + | <h3><a name="section08222016" id="section08222016">08/22/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP</strong> | ||
| + | <br/> </p> | ||
| + | </div> | ||
| + | <h3><a name="section08232016" id="section08232016">08/23/2016</a></h3> | ||
| + | <div class="level4"> | ||
| + | <p> <strong>Sample preparation 9: GOPTS + GFP</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with GFP (1.35mg/ml, volume/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 sectionedit14"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + GFP (1 µl, 10 min) </td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + GFP (2 µl, 10 min) </td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + GFP (5 µl, 10 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + GFP (1 µl, 15 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + GFP (2 µl, 15 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">GOPTS + GFP (5 µl, 15 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">GOPTS + GFP (1 µl, 20 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row8"> | ||
| + | <td class="col0">8.</td> | ||
| + | <td class="col1">GOPTS + GFP (2 µl, 20 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row9"> | ||
| + | <td class="col0">9.</td> | ||
| + | <td class="col1">GOPTS + GFP (5 µl, 20 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row10"> | ||
| + | <td class="col0">10.</td> | ||
| + | <td class="col1">GOPTS (negative control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT14 TABLE [13756-14106] --> | ||
| + | <p> | ||
| + | <br/> <strong>Sample preparation 10: GOPTS + anti-GFP nanobody + - incubated with nanobody (1µl, 330µg/ml, 60 min)</strong> | ||
| + | <br/> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(4%) (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 spores (10µl/spot, 30 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and washed with PBS(1x) (10min, 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) | ||
| + | <br/> </div> | ||
| + | </li> | ||
| + | </ol> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <div class="table sectionedit15"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</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 + GFP (positive control)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + BSA + B53 (negative control)</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <!-- EDIT15 TABLE [14576-14776] --> | ||
| + | <p> | ||
| + | <br/> <strong>Washing test B: GOPTS + anti-GFP nanobody + B53</strong> </p> | ||
| + | <ol> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> incubated with nanobody (1µl, 330µg/ml, 60 min)</div> | ||
| + | </li> | ||
| + | <li class="level1"> | ||
| + | <div class="li"> rinsed and blocked with BSA(4%) (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 spores (10µl/spot, 30 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> | ||
| + | <p> | ||
| + | <br/> </p> | ||
| + | <div class="table sectionedit16"> | ||
| + | <table class="inline"> | ||
| + | <tr class="row0"> | ||
| + | <td class="col0">Spot number</td> | ||
| + | <td class="col1">Sample</td> | ||
| + | <td class="col2">Condition</td> | ||
| + | </tr> | ||
| + | <tr class="row1"> | ||
| + | <td class="col0">1.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">rinsed with PBS(1x) </td> | ||
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0">2.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">swivel with PBS(1x) (0.5 min)</td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0">3.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed with PBS(1x) (1 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0">4.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed with PBS(1x) (2 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row5"> | ||
| + | <td class="col0">5.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed with PBS(1x) (3 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row6"> | ||
| + | <td class="col0">6.</td> | ||
| + | <td class="col1">GOPTS + nanobody + B53</td> | ||
| + | <td class="col2">washed with PBS(1x) (5 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row7"> | ||
| + | <td class="col0">7.</td> | ||
| + | <td class="col1">GOPTS + BSA + B53(negative control)</td> | ||
| + | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row8"> | ||
| + | <td class="col0">8.</td> | ||
| + | <td class="col1">GOPTS + BSA + GFP(negative control)</td> | ||
| + | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row9"> | ||
| + | <td class="col0">9.</td> | ||
| + | <td class="col1">GOPTS + GFP (positive control)</td> | ||
| + | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | <tr class="row10"> | ||
| + | <td class="col0">10.</td> | ||
| + | <td class="col1">GOPTS + B53 (positive control)</td> | ||
| + | <td class="col2">washed with PBS(1x) (10 min, 10 rpm)</td> | ||
| + | </tr> | ||
| + | </table> | ||
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| + | <!-- EDIT16 TABLE [15243-15939] --> | ||
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| + | <br/> </p> | ||
| + | </div> | ||
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| + | <!-- para20 --> | ||
| + | </div> | ||
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| + | </body> | ||
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Revision as of 09:59, 9 October 2016
Lab Journals
Group 4
Members:Kevin, Christian, Vivi, Nathalie
07/04/2016
Preparation of overnight cultures of 5 different Bacillus subtilis strains with GFP on CotZ-fusionprotein
| Nr. | Strain |
| 1. | B52:B. Subtilis w168 amyE::PcgeA-CotZre-gfp-B0014(CM5) |
| 2. | B53:B. Subtilis w168 amyE::PcotYZ-CotZre-gfp-B0014(CM5) |
| 3. | B54:B. Subtilis w168 amyE::PcotYZ-CotZin-gfp-B0014(CM5) |
| 4. | B55:B. Subtilis w168 amyE::PcotV-CotZre-gfp-B0014(CM5) |
| 5. | B56:B. Subtilis w168 amyE::PCotv-CotZin-gfp-B0014(CM5) |
Medium: Preparation of LB-medium with Chloramphenicol: 50µl(1000X) in 50ml LB-Medium
Inoculation: 5 reaction tubes with 5 ml LB-Chloramphenicol-medium have been used. Each has been inoculated with a little piece of filter paper with spores. Cultures have been incubated at 200rpm and 37°C, 16:10Pm to 8am. No growth has been observed !
Further spore production was performed by group 2.
07/05/2016
Preparation of GOPTS coated glass slides
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.
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labeled with a diamond pen
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washed with EtOH then ddH2O
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dried slides with compressed air
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activation of surface with plasma generator
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incubation with GOPTS (overnight)
07/10/2016
Buffer for anti-GFP-nanobody (10 ml)
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61mg Tris
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87mg NaCl
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1ml Glycerin
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2,5 ml Imidazole(1M)
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fill up with ddH2O to 10 ml
07/12/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
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Glass slides were spotted with 0,5 µl nanobody (300µg/ml) (Spot location marked with permanent marker.)
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Slides were incubated over night in the fridge at 4°C in darkness
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150µl BSA(2x) on each spot, 30 min incubation
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washed with PBS(1x)
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washed with 20mM imidazole in PBS(1x)
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washed with PBS(1x)
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washed with ddH2o
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dried with compressed air
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100µl of spore solution on each slide
07/13/2016
Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56
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blocked with BSA(2%) (150µl/spot, 30 min)
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washed with PBS(1x)
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washed with imidazole (20mM) in PBS(1x)
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washed with PBS(1x)
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washed with ddH2O
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dried with compressed air
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incubation with B52-B56 (100µl/spot, 120 min)
-
incubated with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top
| Spot number | 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
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edges sealed with nail polish
Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56
–> pictures inconclusive:pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
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incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min)
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blocked with BSA(2%) (90 min)
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rinsed with PBS(1x)
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stored in PBS(1x) (for 9 days)
| Spot number | 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
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added nanobody to GOPTS plate (300 µg/ml, 5µl)
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tried to pipette rest nanobody down, but didn´t work
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blocked complete slide with BSA(2%)
07/23/016
Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56
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Washed slides with ddH2O
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Add spores (50µl/spot)(B52,B53,B54,B55,B65)
–> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP
Coating of new slides with GOPTS (experiment 3)
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according to protocol
07/24/2016
Preparation of samples 3: B53-B55
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B53, B54, B55 (10µl/spot) on uncoated glass slide
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Mowiol DABCO (5µl/spot)
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covered with cover slip
| Spot number | Sample |
| 1. | GOPTS + nanobody + B52 |
| 2. | GOPTS + nanobody + B53 |
| 3. | GOPTS + nanobody + B54 |
Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56
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fluorescence signal of B53 and B54
Fluorescence microscopy 3 (Olympus 1×71): B53-B55
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fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS
B53 sample: no fluorescence observable (Air bubble)
B54 sample: no fluorescence observable
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d)
07/26/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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Prepared new BSA solution with higher concentration (4% w/v)
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Blocked slides with BSA (4%) ,covered complete slide with BSA
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inkubated over night
07/27/2016
Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with water ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl/spot, overnight)
*Positive controls:
-
Slide with spores B53–> fuorescence observable
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B54 –>Fluorescence observable
| Spot number | Sample |
| 1. | GOPTS + GFP (positive control) |
| 2. | 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) |
Slides of experiment 3
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Washing with PBS
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rinsed with water ddH2O
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dried with compressed air
Positive controls exp. 3:
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GFP direct on GOPTS
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GFP on nanobody
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GFP on BSA blocked surface
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Spores direct on GOPTS
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double concentrated spores direct on glass slide B53 and B54
Spores were added to glass surface and incubated until its dry
07/28/2016
Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54
–>spores appear to not have bound to nanobody
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Washed slides of exp 3 with PBS
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added DABCO (5µl)
07/29/2016
prepared samples for fluorescence microscopy
08/08/2016
Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP
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incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (30 min)
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incubation with B53, B54, B55, B56, GFP (5µl/spot, 60 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
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rinsed with water ddH2O
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dried with compressed air
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incubated with Mowiol DABCO (5µl/spot, overnight)
| Spot number | 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
Test if spores bind Nanobody in solution
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Wt, B53, B54, B56 centrifuged, resuspended in 50 µl nanobody-buffer,
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add 1µl labeled nanobody
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incubated 1h at 4°C
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washed 2 times 10 min with PBS
Problem: no introduction in the confocal microscopy mode
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Flourescence microscopy
Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP
–>only fluorescence detectable on slides 6 and 8
08/10/2016
Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)
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incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C)
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centrifuged (13400 rpm, 2 min)
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washed with PBS(1x) (1 ml)
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repeated steps 2 and 3
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centrifuged (13400 rpm, 2 min)
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resuspended in PBS(1x) (400µl)
| Spot number | 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
Light microscopy Analyse the slides from 28.7.2016 under light mikroscope
08/14/2016
Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54
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incubated with nanobody (1µl, 330µg/ml, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed twice with PBS(1x) (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with spores (10µl/spot, 120 min)
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rinsed and washed twice with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
| Spot number | 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: We want to test if spores (B53) really bind to BSA surface like the previous test showed. We also want to test if the washing procedure has an impact on this result.
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5 slides coated with Gopts
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added 1 µl Nanobody (333µg/ml)
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incubated over night
Washing test A: GOPTS + anti-GFP nanobody + B53
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incubated with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)
08/15/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
Hier Bild von BSA + B53 mit GFP-signal
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rinsed and blocked with BSA(2%) (60 min, 10 rpm)
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rinsed with PBS(1x), washed PBS twice (10 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with B53 (10µl/spot, overnight)
08/16/2016
Washing test A: GOPTS + anti-GFP nanobody + B53
conditions
Coated plates were rinsed with PBS to remove the spore soluten. Slides were put in PBS in Petri dishes and put on the shaker.
| Spot number | 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) |
17/08/2016
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, picture of BSA + B53 must have been a mistake
08/21/2016
Sample preparation 8: GOPTS + anti-GFP nanobody + GFP
–> 10 slides activated with plasmagenerator coated with GOPTS.
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incubated with nanobody (see table below, 60 min)
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rinsed and blocked with BSA(4%) (60 min)
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rinsed and washed with PBS(1x) (5 min, 10 rpm)
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rinsed with ddH2O
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dried with compressed air
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incubated with GFP (see table below, 1.35mg/ml, 20 min)
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rinsed and washed with PBS(1x) (10 min, 10rpm)
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rinsed with ddH20
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dried with compressed air
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incubated with Mowiol DABCO (5µl, overnight)
| Spot number | 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. | GOPTS + BSA + GFP (negative control) |
| 6. | GOPTS + BSA + GFP (negative control) |
Picture after editing of shadow and contrast:
Bedingungen: 5µl,GFP,20min. incubation, mit BSA blocking, 5min. waschen mit PBS,
Blocking Test We want to test if the GOPTS is properly blocked by BSA and if milkprotein is also usefull for blocking
1 Slide with :
| GFP auf GOPTS | GFP good visible , border clearly visible | |
| Milkprotein on GOPTS+GFP | No GFP observable | |
| BSA on GOPTS + GFP | NO GFP observalbe | |
Contamination Test To avoid wrong interpretations of the fluorescence pictures we tested the chemikals and solutions we use, to find out if there are fluorescent contaminations.
| solution | result (GFP LASER) |
| Mowiol/DABCO | a vew fluorecent crystals |
| PBS | a vew fluorecent crystals |
| BSA | no fluorescence |
08/22/2016
Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP
08/23/2016
Sample preparation 9: GOPTS + GFP
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incubated with GFP (1.35mg/ml, volume/incubation: see table below)
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rinsed and blocked with BSA(4%) (30 min)
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rinsed and washed with PBS(1x) (10 min, 10 rpm)
-
rinsed with ddH2O
-
dried with compressed air
-
incubated with Mowiol DABCO (5µl, overnight)
| Spot number | 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 + - incubated 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
-
incubated with Mowiol DABCO (5µl, overnight)
| Spot number | 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
-
incubated 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
-
incubated with Mowiol DABCO (5µl, overnight)
| Spot number | 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) |
