Difference between revisions of "Team:Munich/LabJournal"
| (55 intermediate revisions by 3 users not shown) | |||
| Line 32: | Line 32: | ||
<table width="900" border=0 cellspacing=0 cellpadding=10> | <table width="900" border=0 cellspacing=0 cellpadding=10> | ||
<tr><td colspan=3 align=left valign=center> | <tr><td colspan=3 align=left valign=center> | ||
| − | <font size=7 color=#787878><b>Labbook</b></font> | + | <font size=7 color=#787878><b style="color: #51a7f9">Labbook</b></font> |
</td></tr> | </td></tr> | ||
<tr> | <tr> | ||
<td colspan = 3 align="left"> | <td colspan = 3 align="left"> | ||
<p class="introduction"> | <p class="introduction"> | ||
| − | |||
| − | |||
| − | |||
| − | + | We documented our work chronologically on a daily basis, linked the respective protocols and added all lab results. You can find the entries sorted according to month and sub-project, which can be selected separately. To do actual experiments it is also important to prepare buffer and all kind of stock solutions, agar plates, gels and so on. This basic work is partially not mentioned here, but was also done by the team. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</p> | </p> | ||
| Line 76: | Line 56: | ||
<input type="checkbox" id="Other" name="type" checked="checked" /> <label for="Other">Other</label> | <input type="checkbox" id="Other" name="type" checked="checked" /> <label for="Other">Other</label> | ||
<div class="thirteen wide column" id="labbook-content"> | <div class="thirteen wide column" id="labbook-content"> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<div class="month" id="April" > | <div class="month" id="April" > | ||
| − | + | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<div class="date">Tuesday 25th</div> | <div class="date">Tuesday 25th</div> | ||
| Line 247: | Line 179: | ||
Bacteria lost second plasmid probably. | Bacteria lost second plasmid probably. | ||
</li> | </li> | ||
| − | </ul> | + | </ul> |
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
<div class="month" id="May" > | <div class="month" id="May" > | ||
| − | |||
<div class="date">Tuesday 2nd</div> | <div class="date">Tuesday 2nd</div> | ||
| Line 564: | Line 492: | ||
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
| − | Participants: | + | Participants: Patrick, Julian |
</li> | </li> | ||
<li class="myObservations"> | <li class="myObservations"> | ||
| Line 662: | Line 590: | ||
<img src="https://static.igem.org/mediawiki/2017/a/a5/T--Munich--Results_20170516_Cas13a_Lbu_Heparin_gel.png"> | <img src="https://static.igem.org/mediawiki/2017/a/a5/T--Munich--Results_20170516_Cas13a_Lbu_Heparin_gel.png"> | ||
</li> | </li> | ||
| − | </ul> | + | </ul> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<div class="date">Tuesday 23rd</div> | <div class="date">Tuesday 23rd</div> | ||
<ul class="myEntry cas13a"> | <ul class="myEntry cas13a"> | ||
| Line 698: | Line 619: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="AGAROSEGEL"> | + | <img src="AGAROSEGEL"> |
</li> | </li> | ||
</li> | </li> | ||
| Line 826: | Line 747: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="AGAROSEGEL"> | + | <img src="AGAROSEGEL"> |
</li> | </li> | ||
</li> | </li> | ||
| Line 1,398: | Line 1,319: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 1,459: | Line 1,380: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="PCR image (see 8.6.?)"> | + | <img src="PCR image (see 8.6.?)"> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 1,599: | Line 1,520: | ||
</li> | </li> | ||
<li> | <li> | ||
| − | Cells had OD of 5.97 -> 4.78*10 | + | Cells had OD of 5.97 -> 4.78*10<sup>9</sup> cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 |
</li> | </li> | ||
<li> | <li> | ||
| − | Cells had OD of 5.97 -> 4.78*10 | + | Cells had OD of 5.97 -> 4.78*10<sup>9</sup> cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 </br> |
| − | -> dilluted 1:100 (to V=10mL) and pelleted 209 miL to get to 10 | + | -> dilluted 1:100 (to V=10mL) and pelleted 209 miL to get to 10<sup>7</sup> cells |
</li> | </li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
| − | + | <table class="mediocre-tableEditable-table u-small-spacing-right" style="top: 0px; width: 968px;"><colgroup><col style="width: 45px;"><col data-id="0" style="width: 250px;"><col data-id="1" style="width: 232px;"><col data-id="2" style="width: 216px;"><col data-id="3" style="width: 225px;"></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell u-no-mouse-target"></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">B</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">C</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">D</span></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">1</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-0-0" data-namespace="table-ns3555"><span>Method</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-0-1" data-namespace="table-ns3555"><span>SDS</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-0-2" data-namespace="table-ns3555"><span>NaOH</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-0-3" data-namespace="table-ns3555"><span>TRIzol</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">2</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-1-0" data-namespace="table-ns3555"><span>prepare</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-1-1" data-namespace="table-ns3555"><span>1 mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS -> TE-Buffer + SDS...</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-1-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-1-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">3</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-2-0" data-namespace="table-ns3555"><span>----Lysis</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-2-1" data-namespace="table-ns3555"><span>1x</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-2-2" data-namespace="table-ns3555"><span>3x</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-2-3" data-namespace="table-ns3555"><span>1x</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">4</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-3-0" data-namespace="table-ns3555"><span>separate to tubes Spin 300xG, 5 min ->Ice, TROzol to -20! (100 miL at OD 1 -> 10<sup>7</sup> cells, Max. according to TRIzolPaper</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-3-1" data-namespace="table-ns3555"><span>100 miL bactSusp</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-3-2" data-namespace="table-ns3555"><span>100 miL bact-susp</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-3-3" data-namespace="table-ns3555"><span>100 miL bact-suspension</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">5</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-4-0" data-namespace="table-ns3555"><span>No washing step, bad for mRNA (TRIzolP.)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-4-1" data-namespace="table-ns3555"><span> 300 µl (0.45 ml) TES resuspend pellet</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-4-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-4-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">6</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-5-0" data-namespace="table-ns3555"><span>12,5 uL Inhibitor (40k U/ml, 1 U/miL required)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-5-1" data-namespace="table-ns3555"><span>15 min, 75 °C lysis, vortex</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-5-2" data-namespace="table-ns3555"><span>one probe after another</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-5-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">7</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-6-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-6-1" data-namespace="table-ns3555"><span>(Addition of inhibitor); Transform total volume to gel tube</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-6-2" data-namespace="table-ns3555"><span>resuspend thorougly in 66 µl (100 miL) Solution1</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-6-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">8</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-7-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-7-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-7-2" data-namespace="table-ns3555"><span>132 µl (200 miL) Sol2, INVERT 4x</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-7-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">9</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-8-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-8-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-8-2" data-namespace="table-ns3555"><span>wait 0, 1 ,3 min</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-8-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">10</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-9-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-9-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-9-2" data-namespace="table-ns3555"><span>add 99 µl (150 miL) Sol3, INVERT 4x</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-9-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">11</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-10-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-10-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-10-2" data-namespace="table-ns3555"><span>Total volume: 297 µl (0.45 ml)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-10-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">12</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-11-0" data-namespace="table-ns3555"><span>--phenChloExtr</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-11-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-11-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-11-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">13</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-12-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-12-1" data-namespace="table-ns3555"><span>measure pH, add acid...</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-12-3" data-namespace="table-ns3555"><span>meassure Trizol PH, add acid if needed (pH <5)</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">14</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-13-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-13-1" data-namespace="table-ns3555"><span>add 600 µl [same V (0.45 ml)] Phe:chloro.</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-13-3" data-namespace="table-ns3555"><span>add 1 mL TRIzol to 10<sup>7</sup> cells</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">15</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-14-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-14-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-14-3" data-namespace="table-ns3555"><span>5 min incubation</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">16</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-15-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-15-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-15-3" data-namespace="table-ns3555"><span>add 0.2 mL CHLOROPHORM</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">17</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-16-0" data-namespace="table-ns3555" style="color: rgb(52, 64, 67);"><span>incub 2.5 (2-3) min, RT</span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-16-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-16-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">18</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-17-0" data-namespace="table-ns3555" style="color: rgb(52, 64, 67);"><span>centri 12k g 15 min 4°C</span></td><td class="cell is-text is-alignLeft cell-contents" rowspan="1" colspan="2" data-cid="table-note-part2402-17-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-17-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">19</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-18-0" data-namespace="table-ns3555"><span>angle 45°, extract top phase carefully</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-18-1" data-namespace="table-ns3555"><span>-> discard everything else, prot & DNA</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-18-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-18-3" data-namespace="table-ns3555"><span>-> which Vol for the TRIzol aqueous phase?</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">20</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-19-0" data-namespace="table-ns3555"><span>add 0.4 (0.375) ml Isopropanol, wait 10 min, RT</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-19-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-19-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-19-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">21</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-20-0" data-namespace="table-ns3555"><span>centrifuge 10 min 12k , 4°C -> </span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-20-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-20-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-20-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">22</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-21-0" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span>Aditional step, because RNA pellet was not entierly spinned down: Centrifugation (16000 rcf, 5 min, 4 °C)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-21-1" data-namespace="table-ns3555"><span>big-pellet assay in fridge, but 1. interphase was punctured/gel(DNA?) stuck to pipette 2. s.u.</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-21-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-21-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">23</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-22-0" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span>Aditional step, because suspected RNA fog was not entirely spinned down: remove upper and lower RNA fog phase</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-22-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-22-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-22-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">24</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-23-0" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span>Additional step: Centrifugation (16000 rcf, 5 min, 4 °C)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-23-1" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-23-2" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-23-3" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">25</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-24-0" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span>No RNA pellet or RNA fog any more :(</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-24-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-24-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-24-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">26</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-25-0" data-namespace="table-ns3555" style="color: rgb(225, 69, 22);"><span>Addition of 400 µl isopropanol -> No RNA pellet or fog any more ->prob. removed too much</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-25-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-25-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-25-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">27</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-26-0" data-namespace="table-ns3555"><span>discard supernatant -> RNA in gel-like pellet!</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-26-1" data-namespace="table-ns3555"><span>-> gel hardly visible, nearly draged it out of tube (stuck to pipette...)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-26-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-26-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">28</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-27-0" data-namespace="table-ns3555" style="color: rgb(0, 0, 0);"><span>resuspend in 0.75 ml 70 % (75% ideally) ethanol</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-27-1" data-namespace="table-ns3555"><span>-> storeed RNA at -20°C is stable up to 1 year...</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-27-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-27-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">29</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-28-0" data-namespace="table-ns3555"><span>vortex, centri 5 min 7,5k 4°</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-28-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-28-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-28-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">30</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-29-0" data-namespace="table-ns3555"><span>discard supernatant, air dry for 5-10 min (do NOT let completely dry out)</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-29-1" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-29-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-29-3" data-namespace="table-ns3555"><span></span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">31</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-30-0" data-namespace="table-ns3555"><span>resuspend in RNAse free water 30 µl (20-50 miL) </span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-30-1" data-namespace="table-ns3555"><span>->measurement...</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-30-2" data-namespace="table-ns3555"><span></span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2402-30-3" data-namespace="table-ns3555"><span></span></td></tr></tbody></table> | |
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| Line 1,675: | Line 1,596: | ||
</ul> | </ul> | ||
| − | + | ||
<div class="date">Friday 16th</div> | <div class="date">Friday 16th</div> | ||
| Line 1,726: | Line 1,647: | ||
The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity. | The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity. | ||
</li> | </li> | ||
| − | <img src="A lot of images!!!!"> | + | <img src="A lot of images!!!!"> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 1,747: | Line 1,668: | ||
<ul> | <ul> | ||
<li> | <li> | ||
| − | Cell suspension had OD600 (extrapolated) of 2,220 -> 1.78 x 10 | + | Cell suspension had OD600 (extrapolated) of 2,220 -> 1.78 x 10<sup>9</sup> cells/ml according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158 |
</li> | </li> | ||
<li> | <li> | ||
| − | Experiments were done with 108 and 109 cells per sample, because it failed with 10 | + | Experiments were done with 108 and 109 cells per sample, because it failed with 10<sup>7</sup> cells last time |
</li> | </li> | ||
<li> | <li> | ||
| Line 1,757: | Line 1,678: | ||
<li> | <li> | ||
No pellet after RNA precipitation (-80 °C) and subsequent centrifugation in: | No pellet after RNA precipitation (-80 °C) and subsequent centrifugation in: | ||
| − | <ul> <li>NaOH, 1 min, 10 | + | <ul> <li>NaOH, 1 min, 10<sup>8</sup></li> |
| − | <li>SDS, 10 | + | <li>SDS, 10<sup>9</sup></li> |
| − | <li>Trizol, 10 | + | <li>Trizol, 10<sup>8</sup></li></ul> |
</li> | </li> | ||
<li> Mistakes: | <li> Mistakes: | ||
| Line 1,765: | Line 1,686: | ||
<ul> | <ul> | ||
| − | <li>SDS, 10 | + | <li>SDS, 10<sup>9</sup>: aspirated 100 µl instead of 200 µl supernatant</li> |
| − | <li>NaOH, 10 | + | <li>NaOH, 10<sup>8</sup>, 3 min: contamination with DNA (<10% DNA interphase transfered) </li> |
| − | <li>NaOH, 10 | + | <li>NaOH, 10<sup>9</sup>, 3 min: added + 200 µl of SDS 109 tube</li></ul> |
<li>! Did not calibrate alkaline lysis solution 3 to given pH -> probably more RNA degradation</li> | <li>! Did not calibrate alkaline lysis solution 3 to given pH -> probably more RNA degradation</li> | ||
<li>! Did store RNA at -20 °C instead of -80 -> probably more RNA degradation</li> | <li>! Did store RNA at -20 °C instead of -80 -> probably more RNA degradation</li> | ||
| Line 1,775: | Line 1,696: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | + | <table class="mediocre-tableEditable-table u-small-spacing-right" style="top: 0px; width: 988px;"><colgroup><col style="width: 45px;"><col data-id="0" style="width: 143px;"><col data-id="1" style="width: 100px;"><col data-id="2" style="width: 100px;"><col data-id="3" style="width: 100px;"><col data-id="4" style="width: 100px;"><col data-id="5" style="width: 100px;"><col data-id="6" style="width: 100px;"><col data-id="7" style="width: 100px;"><col data-id="8" style="width: 100px;"></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell u-no-mouse-target"></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">Sample</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">[] [µg/ml]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">adjusted C**</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260/A280</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260/A230</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A230 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A280 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A320 [A]</span></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">1</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-0-0" data-namespace="table-ns3555"><span>SDS, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-1" data-namespace="table-ns3555"><span>36.8</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>55</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-3" data-namespace="table-ns3555"><span>1.704</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-4" data-namespace="table-ns3555"><span>2.044</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-5" data-namespace="table-ns3555"><span>0.047</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-6" data-namespace="table-ns3555"><span>0.094</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-7" data-namespace="table-ns3555"><span>0.056</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-0-8" data-namespace="table-ns3555"><span>0.002</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">2</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-1-0" data-namespace="table-ns3555"><span>SDS, 10<sup>9</sup> cells*</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-1" data-namespace="table-ns3555"><span>119</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>357</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-3" data-namespace="table-ns3555"><span>1.693</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-4" data-namespace="table-ns3555"><span>2.069</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-5" data-namespace="table-ns3555"><span>0.146</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-6" data-namespace="table-ns3555"><span>0.300</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-7" data-namespace="table-ns3555"><span>0.178</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-1-8" data-namespace="table-ns3555"><span>0.002</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">3</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-2-0" data-namespace="table-ns3555"><span>NaOH, 0 min, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-1" data-namespace="table-ns3555"><span>35.2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>53</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-3" data-namespace="table-ns3555"><span>1.725</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-4" data-namespace="table-ns3555"><span>2.146</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-5" data-namespace="table-ns3555"><span>0.042</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-6" data-namespace="table-ns3555"><span>0.089</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-7" data-namespace="table-ns3555"><span>0.052</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-2-8" data-namespace="table-ns3555"><span>0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">4</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-3-0" data-namespace="table-ns3555"><span>NaOH, 0 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-1" data-namespace="table-ns3555"><span>586</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>879</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-3" data-namespace="table-ns3555" style="background-color: rgb(132, 198, 32);"><span>1.923</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-4" data-namespace="table-ns3555"><span>1.993</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-5" data-namespace="table-ns3555"><span>0.740</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-6" data-namespace="table-ns3555"><span>1.470</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-7" data-namespace="table-ns3555"><span>0.767</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-3-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">5</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-4-0" data-namespace="table-ns3555"><span>NaOH, 1 min, 10^8 cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-1" data-namespace="table-ns3555"><span>73.6</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>110</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-3" data-namespace="table-ns3555"><span>1.235</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-4" data-namespace="table-ns3555"><span>0.586</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-5" data-namespace="table-ns3555"><span>0.317</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-6" data-namespace="table-ns3555"><span>0.187</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-7" data-namespace="table-ns3555"><span>0.152</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-4-8" data-namespace="table-ns3555"><span>0.003</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">6</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-5-0" data-namespace="table-ns3555"><span>NaOH, 1 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-1" data-namespace="table-ns3555"><span>370</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>555</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-3" data-namespace="table-ns3555" style="background-color: rgb(132, 198, 32);"><span>1.945</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-4" data-namespace="table-ns3555"><span>1.958</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-5" data-namespace="table-ns3555"><span>0.475</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-6" data-namespace="table-ns3555"><span>0.927</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-7" data-namespace="table-ns3555"><span>0.478</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-5-8" data-namespace="table-ns3555"><span>0.003</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">7</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-6-0" data-namespace="table-ns3555"><span>NaOH, 3 min, 10<sup>8</sup> cells*</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-1" data-namespace="table-ns3555"><span>259</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>389</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-3" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(166, 212, 97);"><span>1.849</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-4" data-namespace="table-ns3555"><span>1.491</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-5" data-namespace="table-ns3555"><span>0.439</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-6" data-namespace="table-ns3555"><span>0.652</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-7" data-namespace="table-ns3555"><span>0.355</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-6-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">8</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-7-0" data-namespace="table-ns3555"><span>NaOH, 3 min, 10<sup>9</sup> cells*</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-1" data-namespace="table-ns3555"><span>275</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>413</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-3" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(166, 212, 97);"><span>1.850</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-4" data-namespace="table-ns3555"><span>2.212</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-5" data-namespace="table-ns3555"><span>0.316</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-6" data-namespace="table-ns3555"><span>0.693</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-7" data-namespace="table-ns3555"><span>0.368</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-7-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">9</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-8-0" data-namespace="table-ns3555"><span>TRIzol, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-1" data-namespace="table-ns3555"><span>194</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>265</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-3" data-namespace="table-ns3555"><span>1.362</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-4" data-namespace="table-ns3555"><span>0.221</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-5" data-namespace="table-ns3555"><span>2.200</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-6" data-namespace="table-ns3555"><span>0.487</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-7" data-namespace="table-ns3555"><span>0.358</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-8-8" data-namespace="table-ns3555"><span>0.002</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">10</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2455-9-0" data-namespace="table-ns3555"><span>TRIzol, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-1" data-namespace="table-ns3555"><span>160</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-2" data-namespace="table-ns3555" style="color: rgb(0, 0, 0); background-color: rgb(246, 217, 77);"><span>218</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-3" data-namespace="table-ns3555"><span>1.429</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-4" data-namespace="table-ns3555"><span>0.488</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-5" data-namespace="table-ns3555"><span>0.822</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-6" data-namespace="table-ns3555"><span>0.403</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-7" data-namespace="table-ns3555"><span>0.283</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2455-9-8" data-namespace="table-ns3555"><span>0.003</span></td></tr></tbody></table> | |
</li><li>Measured [RNA] by Implen Nanophotometer (20170616).</li> | </li><li>Measured [RNA] by Implen Nanophotometer (20170616).</li> | ||
<li>**adjusted for different supernatant V aspirated at extraction step</li> | <li>**adjusted for different supernatant V aspirated at extraction step</li> | ||
| Line 1,948: | Line 1,869: | ||
</ul> | </ul> | ||
| − | + | ||
<div class="date">Friday 23rd</div> | <div class="date">Friday 23rd</div> | ||
| Line 2,241: | Line 2,162: | ||
</li> | </li> | ||
<li> | <li> | ||
| − | + | <table class="mediocre-tableEditable-table u-small-spacing-right" style="top: 0px; width: 1031px;"><colgroup><col style="width: 45px;"><col data-id="0" style="width: 143px;"><col data-id="1" style="width: 143px;"><col data-id="2" style="width: 100px;"><col data-id="3" style="width: 100px;"><col data-id="4" style="width: 100px;"><col data-id="5" style="width: 100px;"><col data-id="6" style="width: 100px;"><col data-id="7" style="width: 100px;"><col data-id="8" style="width: 100px;"></colgroup><tbody><tr><td class="mediocre-tableEditable-axisCell u-no-mouse-target"></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">Sample</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">Number of sample</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">[] [µg/ml]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260/A280</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260/A230</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A230 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A260 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A280 [A]</span></div></td><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--col"><div><span class="mediocre-tableEditable-axisCell-num">A320 [A]</span></div></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">1</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-0-0" data-namespace="table-ns3555"><span>SDS, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-2" data-namespace="table-ns3555"><span>62.4</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-3" data-namespace="table-ns3555"><span>2.080</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-4" data-namespace="table-ns3555"><span>0.118</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-5" data-namespace="table-ns3555"><span>1.343</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-6" data-namespace="table-ns3555"><span>0.174</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-7" data-namespace="table-ns3555"><span>0.093</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-0-8" data-namespace="table-ns3555"><span>0.018</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">2</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-1-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-2" data-namespace="table-ns3555"><span>56.8</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-3" data-namespace="table-ns3555"><span>2.029</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-4" data-namespace="table-ns3555"><span>0.128</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-5" data-namespace="table-ns3555"><span>1.123</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-6" data-namespace="table-ns3555"><span>0.153</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-7" data-namespace="table-ns3555"><span>0.081</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-1-8" data-namespace="table-ns3555"><span>0.011</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">3</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-2-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-2" data-namespace="table-ns3555"><span>47.6</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-3" data-namespace="table-ns3555"><span>1.983</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-4" data-namespace="table-ns3555"><span>0.102</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-5" data-namespace="table-ns3555"><span>1.178</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-6" data-namespace="table-ns3555"><span>0.127</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-7" data-namespace="table-ns3555"><span>0.068</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-2-8" data-namespace="table-ns3555"><span>0.008</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">4</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-3-0" data-namespace="table-ns3555"><span>SDS, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-2" data-namespace="table-ns3555"><span>354</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-3" data-namespace="table-ns3555"><span>1.782</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-3-4" data-namespace="table-ns3555"><span>>0.4</span></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-3-5" data-namespace="table-ns3555"><span>>2.5</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-6" data-namespace="table-ns3555"><span>0.913</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-7" data-namespace="table-ns3555"><span>0.525</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-3-8" data-namespace="table-ns3555"><span>0.029</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">5</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-4-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-2" data-namespace="table-ns3555"><span>260</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-3" data-namespace="table-ns3555"><span>1.857</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-4" data-namespace="table-ns3555"><span>0.295</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-5" data-namespace="table-ns3555"><span>2.222</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-6" data-namespace="table-ns3555"><span>0.665</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-7" data-namespace="table-ns3555"><span>0.365</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-4-8" data-namespace="table-ns3555"><span>0.015</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">6</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-5-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-2" data-namespace="table-ns3555"><span>376</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-3" data-namespace="table-ns3555"><span>1.801</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-4" data-namespace="table-ns3555"><span>0.392</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-5" data-namespace="table-ns3555"><span>2.422</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-6" data-namespace="table-ns3555"><span>0.962</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-7" data-namespace="table-ns3555"><span>0.544</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-5-8" data-namespace="table-ns3555"><span>0.022</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">7</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-6-0" data-namespace="table-ns3555"><span>NaOH, 0 min, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-2" data-namespace="table-ns3555"><span>39.2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-3" data-namespace="table-ns3555" style="color: rgb(0, 0, 0);"><span>1.690</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-4" data-namespace="table-ns3555"><span>2.042</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-5" data-namespace="table-ns3555"><span>0.056</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-6" data-namespace="table-ns3555"><span>0.106</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-7" data-namespace="table-ns3555"><span>0.066</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-6-8" data-namespace="table-ns3555"><span>0.008</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">8</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-7-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-2" data-namespace="table-ns3555"><span>53.6</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-3" data-namespace="table-ns3555" style="color: rgb(0, 0, 0);"><span>1.614</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-4" data-namespace="table-ns3555"><span>1.887</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-5" data-namespace="table-ns3555"><span>0.086</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-6" data-namespace="table-ns3555"><span>0.149</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-7" data-namespace="table-ns3555"><span>0.098</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-7-8" data-namespace="table-ns3555"><span>0.015</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">9</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-8-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-2" data-namespace="table-ns3555"><span>68.8</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-3" data-namespace="table-ns3555"><span>1.720</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-4" data-namespace="table-ns3555"><span>2.457</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-5" data-namespace="table-ns3555"><span>0.070</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-6" data-namespace="table-ns3555"><span>0.172</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-7" data-namespace="table-ns3555"><span>0.100</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-8-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">10</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-9-0" data-namespace="table-ns3555"><span>NaOH, 0 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-2" data-namespace="table-ns3555"><span>629</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-3" data-namespace="table-ns3555"><span>1.911</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-4" data-namespace="table-ns3555"><span>2.466</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-5" data-namespace="table-ns3555"><span>0.643</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-6" data-namespace="table-ns3555"><span>1.578</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-7" data-namespace="table-ns3555"><span>0.828</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-9-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">11</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-10-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-2" data-namespace="table-ns3555" style="background-color: rgb(249, 220, 220);"><span>17.6</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-3" data-namespace="table-ns3555"><span>1.692</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-4" data-namespace="table-ns3555"><span>2.200</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-5" data-namespace="table-ns3555"><span>0.019</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-6" data-namespace="table-ns3555"><span>0.043</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-7" data-namespace="table-ns3555"><span>0.025</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-10-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">12</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-11-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-2" data-namespace="table-ns3555"><span>619</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-3" data-namespace="table-ns3555"><span>1.897</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-4" data-namespace="table-ns3555"><span>2.453</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-5" data-namespace="table-ns3555"><span>0.636</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-6" data-namespace="table-ns3555"><span>1.553</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-7" data-namespace="table-ns3555"><span>0.821</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-11-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">13</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-12-0" data-namespace="table-ns3555"><span>NaOH, 1 min, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-2" data-namespace="table-ns3555"><span>52</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-3" data-namespace="table-ns3555"><span>1.806</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-4" data-namespace="table-ns3555"><span>2.203</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-5" data-namespace="table-ns3555"><span>0.059</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-6" data-namespace="table-ns3555"><span>0.130</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-7" data-namespace="table-ns3555"><span>0.072</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-12-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">14</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-13-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-2" data-namespace="table-ns3555"><span>33.2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-3" data-namespace="table-ns3555"><span>1.729</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-4" data-namespace="table-ns3555"><span>2.371</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-5" data-namespace="table-ns3555"><span>0.035</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-6" data-namespace="table-ns3555"><span>0.083</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-7" data-namespace="table-ns3555"><span>0.048</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-13-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">15</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-14-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-2" data-namespace="table-ns3555"><span>58.4</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-3" data-namespace="table-ns3555"><span>1.738</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-4" data-namespace="table-ns3555"><span>2.393</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-5" data-namespace="table-ns3555"><span>0.061</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-6" data-namespace="table-ns3555"><span>0.146</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-7" data-namespace="table-ns3555"><span>0.084</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-14-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">16</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-15-0" data-namespace="table-ns3555"><span>NaOH, 1 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-2" data-namespace="table-ns3555"><span>606</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-3" data-namespace="table-ns3555"><span>1.893</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-4" data-namespace="table-ns3555"><span>2.469</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-5" data-namespace="table-ns3555"><span>0.620</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-6" data-namespace="table-ns3555"><span>1.522</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-7" data-namespace="table-ns3555"><span>0.807</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-15-8" data-namespace="table-ns3555"><span>0.006</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">17</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-16-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-2" data-namespace="table-ns3555"><span>627</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-3" data-namespace="table-ns3555"><span>1.898</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-4" data-namespace="table-ns3555"><span>2.431</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-5" data-namespace="table-ns3555"><span>0.651</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-6" data-namespace="table-ns3555"><span>1.574</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-7" data-namespace="table-ns3555"><span>0.832</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-16-8" data-namespace="table-ns3555"><span>0.006</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">18</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-17-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-2" data-namespace="table-ns3555" style="background-color: rgb(249, 220, 220);"><span>22.4</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-3" data-namespace="table-ns3555"><span>1.806</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-4" data-namespace="table-ns3555"><span>2.435</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-5" data-namespace="table-ns3555"><span>0.023</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-6" data-namespace="table-ns3555"><span>0.056</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-7" data-namespace="table-ns3555"><span>0.031</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-17-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">19</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-18-0" data-namespace="table-ns3555"><span>NaOH, 3 min, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-2" data-namespace="table-ns3555"><span>44</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-3" data-namespace="table-ns3555"><span>1.864</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-4" data-namespace="table-ns3555"><span>2.444</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-5" data-namespace="table-ns3555"><span>0.044</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-6" data-namespace="table-ns3555"><span>0.109</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-7" data-namespace="table-ns3555"><span>0.058</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-18-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">20</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-19-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-2" data-namespace="table-ns3555"><span>61.2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-3" data-namespace="table-ns3555"><span>1.779</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-4" data-namespace="table-ns3555"><span>2.593</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-5" data-namespace="table-ns3555"><span>0.058</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-6" data-namespace="table-ns3555"><span>0.152</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-7" data-namespace="table-ns3555"><span>0.085</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-19-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">21</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-20-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-2" data-namespace="table-ns3555" style="background-color: rgb(249, 220, 220);"><span>5.6</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-3" data-namespace="table-ns3555"><span>1.400</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-4" data-namespace="table-ns3555"><span>2.800</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-5" data-namespace="table-ns3555"><span>0.004</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-6" data-namespace="table-ns3555"><span>0.013</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-7" data-namespace="table-ns3555"><span>0.009</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-20-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">22</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-21-0" data-namespace="table-ns3555"><span>NaOH, 3 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-2" data-namespace="table-ns3555"><span>619</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-3" data-namespace="table-ns3555"><span>1.908</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-4" data-namespace="table-ns3555"><span>2.432</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-5" data-namespace="table-ns3555"><span>0.643</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-6" data-namespace="table-ns3555"><span>1.554</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-7" data-namespace="table-ns3555"><span>0.818</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-21-8" data-namespace="table-ns3555"><span>0.007</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">23</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-22-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-2" data-namespace="table-ns3555"><span>570</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-3" data-namespace="table-ns3555"><span>1.927</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-4" data-namespace="table-ns3555"><span>2.468</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-5" data-namespace="table-ns3555"><span>0.583</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-6" data-namespace="table-ns3555"><span>1.430</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-7" data-namespace="table-ns3555"><span>0.745</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-22-8" data-namespace="table-ns3555"><span>0.006</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">24</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-23-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-2" data-namespace="table-ns3555" style="background-color: rgb(249, 220, 220);"><span>20.4</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-3" data-namespace="table-ns3555"><span>1.759</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-4" data-namespace="table-ns3555"><span>2.429</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-5" data-namespace="table-ns3555"><span>0.020</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-6" data-namespace="table-ns3555"><span>0.050</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-7" data-namespace="table-ns3555"><span>0.028</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-23-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">25</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-24-0" data-namespace="table-ns3555"><span>NaOH, 10 min, 10<sup>8</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-2" data-namespace="table-ns3555"><span>10.8</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-3" data-namespace="table-ns3555"><span>1.588</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-4" data-namespace="table-ns3555"><span>2.250</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-5" data-namespace="table-ns3555"><span>0.012</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-6" data-namespace="table-ns3555"><span>0.027</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-7" data-namespace="table-ns3555"><span>0.017</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-24-8" data-namespace="table-ns3555"><span>0.000</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">26</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-25-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-2" data-namespace="table-ns3555"><span>8.8</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-3" data-namespace="table-ns3555"><span>1.571</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-4" data-namespace="table-ns3555"><span>3.143</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-5" data-namespace="table-ns3555"><span>0.006</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-6" data-namespace="table-ns3555"><span>0.021</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-7" data-namespace="table-ns3555"><span>0.013</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-25-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">27</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-26-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-2" data-namespace="table-ns3555"><span>4.4</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-3" data-namespace="table-ns3555"><span>1.375</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-4" data-namespace="table-ns3555"><span>3.667</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-5" data-namespace="table-ns3555"><span>0.002</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-6" data-namespace="table-ns3555"><span>0.010</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-7" data-namespace="table-ns3555"><span>0.007</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-26-8" data-namespace="table-ns3555"><span>-0.001</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">28</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-27-0" data-namespace="table-ns3555"><span>NaOH, 10 min, 10<sup>9</sup> cells</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-1" data-namespace="table-ns3555"><span>1</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-2" data-namespace="table-ns3555"><span>503</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-3" data-namespace="table-ns3555"><span>1.916</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-4" data-namespace="table-ns3555"><span>2.499</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-5" data-namespace="table-ns3555"><span>0.508</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-6" data-namespace="table-ns3555"><span>1.262</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-7" data-namespace="table-ns3555"><span>0.661</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-27-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">29</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-28-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-1" data-namespace="table-ns3555"><span>2</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-2" data-namespace="table-ns3555"><span>564</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-3" data-namespace="table-ns3555"><span>1.911</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-4" data-namespace="table-ns3555"><span>2.487</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-5" data-namespace="table-ns3555"><span>0.572</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-6" data-namespace="table-ns3555"><span>1.415</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-7" data-namespace="table-ns3555"><span>0.743</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-28-8" data-namespace="table-ns3555"><span>0.005</span></td></tr><tr><td class="mediocre-tableEditable-axisCell mediocre-tableEditable-axisCell--row"><div><span class="mediocre-tableEditable-axisCell-num">30</span></div></td><td class="cell is-text is-alignLeft cell-contents" data-cid="table-note-part2640-29-0" data-namespace="table-ns3555"><span></span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-1" data-namespace="table-ns3555"><span>3</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-2" data-namespace="table-ns3555"><span>611</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-3" data-namespace="table-ns3555"><span>1.902</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-4" data-namespace="table-ns3555"><span>2.467</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-5" data-namespace="table-ns3555"><span>0.625</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-6" data-namespace="table-ns3555"><span>1.533</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-7" data-namespace="table-ns3555"><span>0.809</span></td><td class="cell is-value is-alignRight cell-contents" data-cid="table-note-part2640-29-8" data-namespace="table-ns3555"><span>0.006</span></td></tr></tbody></table> | |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 2,359: | Line 2,280: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GELIMAGEs.5.7."> | + | <img src="GELIMAGEs.5.7."> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 2,444: | Line 2,365: | ||
</ul> | </ul> | ||
| − | + | ||
<div class="date">Friday 7th</div> | <div class="date">Friday 7th</div> | ||
| Line 2,469: | Line 2,390: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 2,529: | Line 2,450: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="graph"> | + | <img src="graph"> |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 2,546: | Line 2,467: | ||
</li> | </li> | ||
| − | </ul | + | </ul> |
<div class="date">Friday 14th</div> | <div class="date">Friday 14th</div> | ||
| Line 2,588: | Line 2,509: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GRAPH"> | + | <img src="GRAPH"> |
</li> | </li> | ||
</ul> | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RNA Extraction with commercial kit </b> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Patrick | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | used ReliaPrep miRNA Cell and Tissue Miniprep System (Promega) according to instructions | ||
| + | </li> | ||
| + | <li> | ||
| + | Specification: Use in range of 10<sup>2</sup> to 10<sup>6</sup> cells. Tested 10<sup>6</sup> and 10<sup>8</sup> cells | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | No nanodrop-measureable amount of RNA/DNA with 10<sup>6</sup> cell, <10 ug/ml RNA with 10<sup>8</sup> cells | ||
| + | Terrible yield, repeat again to be sure | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Monday 17th</div> | <div class="date">Monday 17th</div> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 2,611: | Line 2,557: | ||
<img src="https://static.igem.org/mediawiki/2017/0/0c/T--Munich--Results_20170717_Activity_Assay_results.png"> | <img src="https://static.igem.org/mediawiki/2017/0/0c/T--Munich--Results_20170717_Activity_Assay_results.png"> | ||
</li> | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RNA Extraction with commercial kit </b> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Patrick | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | used ReliaPrep miRNA Cell and Tissue Miniprep System (Promega) according to instructions | ||
| + | </li> | ||
| + | <li> | ||
| + | Specification: Use in range of 10<sup>2</sup> to 10<sup>6</sup> cells. Tested 10<sup>6</sup> and 10<sup>8</sup> cells | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Again no measureable amount of RNA for 10<sup>6</sup> cells. | ||
| + | For 10<sup>8</sup> cells, RNA concentration was increased through column-binding and elution (small elution volume) | ||
| + | Elution seems effective, second elution has only 20 % of first elution's amount of RNA | ||
| + | </li> | ||
</ul> | </ul> | ||
<div class="date">Tuesday 18th</div> | <div class="date">Tuesday 18th</div> | ||
| Line 2,722: | Line 2,694: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GELIMAGE maybe in next days"> | + | <img src="GELIMAGE maybe in next days"> |
</li> | </li> | ||
| Line 2,797: | Line 2,769: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
| Line 2,850: | Line 2,822: | ||
<div class="month" id="August" > | <div class="month" id="August" > | ||
<div class="date firstOfMonth">Tuesday 1st</div> | <div class="date firstOfMonth">Tuesday 1st</div> | ||
| − | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">DNA Gel-Purification and Genomic DNA Purification</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Purification of b-Gal fragments from 1 % Agarose Gel <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 60 µl per lane | ||
| + | </li> | ||
| + | Genomic DNA purification from E.coli W3110+ | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | DNA was lost during the purification | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Wednesday 2nd</div> | <div class="date">Wednesday 2nd</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and PCR-Cleanup kit</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Repeated PCR of beta-Gal N and C Terminal fragments from 31.7. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 3 different Templates: | ||
| + | <ul> | ||
| + | <li> | ||
| + | W3110 genomic DNA | ||
| + | </li> | ||
| + | <li> | ||
| + | W3110 genomic DNA diluted 1:100 | ||
| + | </li> | ||
| + | <li> | ||
| + | W3110 2 µl of cryostock | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li> | ||
| + | Clean-up of PCR with Purification Kit and elution in 33 µl nf water | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 2,887: | Line 2,929: | ||
</ul> | </ul> | ||
<div class="date">Thursday 3rd</div> | <div class="date">Thursday 3rd</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Gel of purified PCR <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 1,5% small Gel, 120 V, 30 minutes | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--Results_20170803_Max_Gel.png"> | ||
| + | <ul> | ||
| + | <li>PCR with undiluted extracted DNA didn't work.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| − | |||
<div class="date">Monday 7th</div> | <div class="date">Monday 7th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Protein-Purifcation of Lbu and Lsh</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Ludwig, Teeradon <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Inoculation of main cultures of Rosetta p2CT-His-MBP-Lbu_C13a_WT and Lsh in LB(Carb+Cm) <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 2,917: | Line 3,015: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 8th</div> | <div class="date">Tuesday 8th</div> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>First test with 3D printed 96-well plate</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Tested different concentrations of fluorescein in a 3D printed 96 well plate on paper. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Scanned at differnt gains at a constant focal height. | ||
| + | </li> | ||
| + | <li> | ||
| + | Dilutions were 1:20; 1:400 and 1:8000 of Fluorescein-FITC in water | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | No light bleeding was observed in the experiment | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 2,946: | Line 3,077: | ||
<div class="date">Wednesday 9th</div> | <div class="date">Wednesday 9th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Expression and Harvesting of Cas13a variants</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Ludwig <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Harvested Cas13a Lbu overexpression. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Inoculated 2 litres 2xYT media with Rosetta Cas13a Lsh and let them grow at 16 °C overnight with 1 mM IPTG | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 2,982: | Line 3,139: | ||
<div class="date">Thursday 10th</div> | <div class="date">Thursday 10th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>His Purification of Cas13a Lbu and Harvesting of Cas13a Lsh</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Ludwig, Milica, Sven, Benedikt <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | His purification of Cas13a Lbu according to protocol. | ||
| + | </li> | ||
| + | <li> | ||
| + | Elution in 4 times 4 ml. | ||
| + | </li> | ||
| + | <li> | ||
| + | ON Dialysis with TEV protease in 2 litres of Lysis buffer. | ||
| + | </li> | ||
| + | <li> | ||
| + | Harvesting of Cas13a Lsh according to protocol | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,047: | Line 3,235: | ||
</ul> | </ul> | ||
<div class="date">Friday 11th</div> | <div class="date">Friday 11th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Reverse HIS purification and SEC of Cas13a Lbu</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification and SEC protein purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Ludwig, Benedikt, Max, Sven <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Incubation of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, 4 °C) <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Application on column, keep FT (there is our protein) | ||
| + | </li> | ||
| + | <li> | ||
| + | Elution of His-MBP with ca. 10 mL Elution buffer (for gel analysis) | ||
| + | </li> | ||
| + | <li> | ||
| + | Washing of Ni-NTA bead (3x with water) and storage in 20 % EtOH | ||
| + | </li> | ||
| + | <li> | ||
| + | Up-concentration of Cas13a Lbu and buffer exchange in SEC buffer (centrifugal filter: MWCO: 30 kDa, 4 °C, 5000 RCF) | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | See Gel image of 14.8. for results. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| − | <b> | + | <b>o</b> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
| Line 3,110: | Line 3,338: | ||
</ul> | </ul> | ||
<div class="date">Monday 14th</div> | <div class="date">Monday 14th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>SDS PAGE of Lbu purifcations</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Ludwig <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | SDS-PAGE of Lbu purification from last week <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,161: | Line 3,419: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 16th</div> | <div class="date">Wednesday 16th</div> | ||
| − | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | |
| + | <li class="myTitel"> | ||
| + | <b>Cas13a preparation</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Benedikt, Ludwig <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Concentration measurment of Lbu, Lsh and Lwa with Bradford essay <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | Concentration of proteins measured(µg/ml): | ||
| + | <li> | ||
| + | 718 - Lbu 13.6 (pooled fractions 6, 7, 8) | ||
| + | </li> | ||
| + | <li> | ||
| + | 387 - Lbu 12.7 | ||
| + | </li> | ||
| + | <li> | ||
| + | 90,8 - Lsh 13.6 (pooled fraction 7,8,9) | ||
| + | </li> | ||
| + | <li> | ||
| + | 379 -Lwa 5.7 (elution 1) | ||
| + | </li> | ||
| + | <li> | ||
| + | 1620 - Lwa 5.7 (elution 2) | ||
| + | </li> | ||
| + | <li> | ||
| + | 818 - Lwa 5.7 (elution 3) | ||
| + | </li> | ||
| + | <li> | ||
| + | 405 -Lwa 5.7 (elution 4) | ||
| + | </li> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Thursday 17th</div> | <div class="date">Thursday 17th</div> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 3,189: | Line 3,500: | ||
<img src="https://static.igem.org/mediawiki/2017/6/61/T--Munich--Results_20170817_DNA_RnA_Annealing_Results.png"> | <img src="https://static.igem.org/mediawiki/2017/6/61/T--Munich--Results_20170817_DNA_RnA_Annealing_Results.png"> | ||
</li> | </li> | ||
| − | </ul> | + | </ul> |
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Protein-Expression of Lbu and Lwa</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein-Expression</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Benedikt, Ludwig, Milica <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Expression of Rosetta Cas13a Lbu (3L 2xYT Medium, 16°C ON) <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Expression of Rosetta Cas13a Lwa (2L 2xYT Medium, 16°C ON) | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Friday 18th</div> | <div class="date">Friday 18th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Harvesting and Purification of Cas13a Lbu and Lsh</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Benedikt, Milica <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Harvesting of both expressions <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Purification of Cas13a Lbu according to protocol. | ||
| + | </li> | ||
| + | <li> | ||
| + | Elution in 4 times 3 ml elution buffer. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Sunday 20th</div> | <div class="date">Sunday 20th</div> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 3,212: | Line 3,581: | ||
</ul> | </ul> | ||
<div class="date">Monday 21st</div> | <div class="date">Monday 21st</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Continuation of Cas13a purification</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Benedikt, Milica, Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Continued His purification of Cas13a Lbu <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Overnight dialysis with TEV protesae | ||
| + | </li> | ||
| + | |||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/fe/T--Munich--Results_20170821_Max_Gel.png"> | ||
| + | <ul> | ||
| + | <li>As visible on the gel the elutions 1 and 2 contained the highest amount of protein and were pooled for the TEV cleavage.</li> | ||
| + | </ul> <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,293: | Line 3,697: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 22nd</div> | <div class="date">Tuesday 22nd</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Reverse His purification of Cas13a Lbu</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Benedikt, Milica <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Incubation (shaker!!) of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, ice) <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Application on column, keep FT (there is our protein) (on ice) | ||
| + | </li> | ||
| + | <li> | ||
| + | Washing with 10 mL lysis-buffer | ||
| + | </li> | ||
| + | </li> | ||
| + | <li> | ||
| + | Elution of His-MBP with ca. 10 mL Elution buffer (for gel analysis) | ||
| + | </li> | ||
| + | <li> | ||
| + | Washing of Ni-NTA bead (3x5mL with water) and storage in 20 % EtOH | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,377: | Line 3,821: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 23rd</div> | <div class="date">Wednesday 23rd</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Cas13a purification</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Size Exclusion Chromatography</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Benedikt, Milica <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Measuring concentration after Reverse His Lbu <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Upconcentration and buffer exchange of Lbu in 30 kDa Centricon | ||
| + | </li> | ||
| + | <li> | ||
| + | SEC Run of Lbu according to protcol | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | See SDS PAGE of 24.8. to see the SEC result. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,440: | Line 3,917: | ||
</ul> | </ul> | ||
<div class="date">Thursday 24th</div> | <div class="date">Thursday 24th</div> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>SDS-Gel of SEC-Run from 23.8.</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Chris <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | 10% SDS PAGE of SEC Run samples <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Marker; Sample prior to run; Fractions 5-16; FT Centricon | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/31/T--Munich--Results_20170824_Max_Gel.png"> | ||
| + | <ul><li>The samples with the highest protein concentration were pooled and used for further experiments.</li></ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,686: | Line 4,195: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 29th</div> | <div class="date">Tuesday 29th</div> | ||
| + | |||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5 Polymerase, PCR Cleanup Kit, Restriction digest and Ligation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Resuspension of gblocks Split_Uni C and Split_Uni N at a conc. of 100 nMol | ||
| + | </li> | ||
| + | <li> | ||
| + | PCR of SPlit Uni C und Split Uni N with Q5 Polymerase <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Clean-up with Monarch Kit | ||
| + | </li> | ||
| + | <li> | ||
| + | Digestion of PCR products and psB1C3 with PstI-HF und EcoRI-HF in Cutsmart buffer | ||
| + | </li> | ||
| + | <li> | ||
| + | Dephosphorilation with Antarctic Phosphatase | ||
| + | </li> | ||
| + | <li> | ||
| + | Ligation ON with T4 DNA Ligase | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,853: | Line 4,401: | ||
</ul> | </ul> | ||
<div class="date">Thursday 31st</div> | <div class="date">Thursday 31st</div> | ||
| − | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | E. comp transformation of E. coli Turbo with 1 µl of overnight ligation of 27.8. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Platted on Cm plates overnight. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,891: | Line 4,463: | ||
<div class="month" id="September" > | <div class="month" id="September" > | ||
<div class="date firstOfMonth">Friday 1st</div> | <div class="date firstOfMonth">Friday 1st</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Ligation</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Plates showed no colonies, as there wasn't enough backbone used. | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Repeated digestion and Ligation from Thursday 31.8 and ligated 1 C-Terminal and 3 N-Terminal fragments respectively. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry target"> | <ul class="myEntry target"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 3,917: | Line 4,515: | ||
</ul> | </ul> | ||
| − | |||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
| Line 3,960: | Line 4,555: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Nukleotide-silica binding with different buffers</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa per sample | ||
| + | </li> | ||
| + | <li> | ||
| + | Did NOT wash silica particles before with binding buffer (pH) and NOT during procedure with ethanol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Both buffers show terrible A260/280 ration, need washing step and need to adjust for acidic silica-solution | ||
| + | </br><img style="width:auto;height:auto;max-width:100%;" src="https://static.igem.org/mediawiki/2017/5/55/T--Munich--pic--rnaextraction_WOwashing.png" > | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
<div class="date">Saturday 2nd</div> | <div class="date">Saturday 2nd</div> | ||
| − | + | <ul class="myEntry readout"> | |
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Chem_Trafo</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian, Milica, Jorge | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS. All | ||
| + | </li> | ||
| + | <li> | ||
| + | Lysozyme digestion: According to protocol. | ||
| + | </li> | ||
| + | <li> | ||
| + | Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17 for gel picture. | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Nukleotide-silica binding with different buffers</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa | ||
| + | </li> | ||
| + | <li> | ||
| + | Washed according to protocol | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Acetate-buffer results acceptable now, GuSCN-buffer still a problem.GuSCN probably too difficult clean up, needs too many washing steps. | ||
| + | </br> <img style="width:auto;height:auto;max-width:100%;" src="https://static.igem.org/mediawiki/2017/a/a5/T--Munich--pic--rnaextraction_Wwashing.png" > | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <div class="date">Monday 4th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Plates showed no colonies as there was no SOC step. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Retransformation of Ligation from Friday into E.Coli Turbo e. comp. | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,004: | Line 4,723: | ||
See results Urea-SDS-PAGE from 05.09.17. | See results Urea-SDS-PAGE from 05.09.17. | ||
</li> | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Nukleotide-silica binding with different buffers</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Did NOT incubate silica with acetate buffer for a day, prepared it fresh | ||
| + | </li> | ||
| + | <li> | ||
| + | Used 10 ul of RNA-solution from Aug 31 RNA-extraction by Dawa | ||
| + | </li> | ||
| + | <li> | ||
| + | Washed according to protocol, but did NOT wait a day for silica-buffer interaction with Acetate-buffer | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Terrible yield, need buffer-silica incubation in protocol. | ||
| + | </br> <img style="width:auto;height:auto;max-width:100%;" src="https://static.igem.org/mediawiki/2017/1/18/T--Munich--pic--rnaextraction_WOprep.png" > | ||
| + | </li> | ||
</ul> | </ul> | ||
<div class="date">Tuesday 5th</div> | <div class="date">Tuesday 5th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>DNA Amplification Test using Recombinase Polymerase Amplification </b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | DNA Amplification with RPA was tested using the provided Control Reaction by TwistDx and our BioBrick His6-TEV plasmid | ||
| + | incubation times of 40 minutes for Control and Sample 1 and 60 minutes for sample 2. Differences due to incubation time | ||
| + | was tested. | ||
| + | </li> | ||
| + | <li> | ||
| + | RPA according to protocol | ||
| + | </li> | ||
| + | <li> | ||
| + | PCR cleanup according to protocol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | |||
| + | <ul> | ||
| + | <li>Positive Control worked.</li> | ||
| + | <li>RPA of TEV could not produce the whole (~1300 bp) construct but would produce amplicons of shorter size (100-500 bp) which was anticipated since TwistDx protocol says optimal amplicon length is 500 bp.</li> | ||
| + | <li>Full length double-stranded DNA could consequently not be formed.</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/24/T--Munich--Results_20170905_test_RPA_Cy2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Ran a small 1.75% TAE-Agarose Gel at 150 V for 50 minutes and post stained with Sybr Gold <!--All--> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | <li class="myTitel"> | ||
| + | <b>Overnight Ligation of Intein-Extein-Construct</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Ligation of DNA-Fragments</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Overnight ligation in ice bath with psb1C3 and psb4A5 as backbones and a 3:1 molar excess of insert <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 200 ng backbone | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Preparation of chemical competent Turbo cells</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Preparation of chemical competent cells</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Plated one aliquot as negative controll <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | No colonies on plates, therefore usefull competent cells. <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,098: | Line 4,966: | ||
<div class="date">Wednesday 6th</div> | <div class="date">Wednesday 6th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNaseAlert Experiment with E.coli RNA with different concentrations of target E.coli RNA </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Jorge, Igor | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Run1: Amount of RNA: 1 nm Cas13a, 10 nm crRNA | ||
| + | </li> | ||
| + | <li> | ||
| + | Run2: Amount of RNA: 1 nm Cas13a, 10 nm crRNA | ||
| + | </li> | ||
| + | <li> | ||
| + | img src="https://static.igem.org/mediawiki/2017/b/be/T--Munich--pic--platereader_pipScheme_6_09_Ecoli.png" > | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Chem and electro Transformation</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation into chem and e. competent E.coli Turbo and DH5 alpha cells. <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | No colonies in the evening, therefore the plates were left at room temperature overnight. <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,123: | Line 5,045: | ||
</ul> | </ul> | ||
<div class="date">Thursday 7th</div> | <div class="date">Thursday 7th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>RNA Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Picked 128 colonies from plates of 06.09.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 4 strip tubes of each construct( psB1C3_C; psB1c3_N; psB4A5_c; psB4A5_N) | ||
| + | </li> | ||
| + | <li> | ||
| + | Colony PCR with OneTaq Polymerase | ||
| + | </li> | ||
| + | <li> | ||
| + | Samples loaded onto monster Gel (Samples order is interlaced) | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Results_20170907_Max_Results.png"> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,167: | Line 5,125: | ||
<ul> | <ul> | ||
<li> | <li> | ||
| − | Diluted E. coli culture to 10 | + | Diluted E. coli culture to 10<sup>8</sup> cells/ml, used 300 uL as sample |
</li> | </li> | ||
<li> | <li> | ||
| Line 4,177: | Line 5,135: | ||
<h4>Results</h4> | <h4>Results</h4> | ||
Hardly any RNA detectable (concentration < 10 ug/ml). Abysmal 260/280-ratio </br> | Hardly any RNA detectable (concentration < 10 ug/ml). Abysmal 260/280-ratio </br> | ||
| − | -> Heat-only lysis is too inefficient for that low cell counts, use 10 | + | -> Heat-only lysis is too inefficient for that low cell counts, use 10<sup>9</sup> next time. |
</li> | </li> | ||
</ul> | </ul> | ||
| Line 4,183: | Line 5,141: | ||
<div class="date">Monday 11th</div> | <div class="date">Monday 11th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Precultures of 16 colonies from plates of 06.09.17. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Precultures in 5 ml LB with corresponding antibiotic | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | |||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,205: | Line 5,191: | ||
<div class="date">Tuesday 12th</div> | <div class="date">Tuesday 12th</div> | ||
| − | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prep with Qiagen Kit and Sequencing Sample preparation </a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Mini Prep of precultures from 11.09.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Send all 16 plasmids for sequencing with primer VR | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | psB4A5_C-Terminal 1-7 and 3-4 were positive.<!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Wednesday 13th</div> | <div class="date">Wednesday 13th</div> | ||
| − | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest and PCR protocol with Q5 polymerase</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Send Samples psB4A5_C-Terminal 1-7 and 3-4 for sequencing with primer VF <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | New digestion of unamplified N-Terminal g-block (200 ng in 50 µl with EcoRI-HF and SpeI-HF) | ||
| + | </li> | ||
| + | <li> | ||
| + | PCR of psB1C3 with psb1A3_fw and psb1A3_rev , to get an opened backbone (Q5 polymerase protocol with 66 °C as annealing temerature and 1 minute elongation time) | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | PsB4A5_C terminal 1-7 was sequenced completly and used for further cloning steps. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,224: | Line 5,272: | ||
<ul> | <ul> | ||
<li> | <li> | ||
| − | Lysis at 90°C, used 300 uL of E. coli suspension diluted to 2*10 | + | Lysis at 90°C, used 300 uL of E. coli suspension diluted to 2*10<sup>9</sup> cells/ml |
</li> | </li> | ||
<li> | <li> | ||
| Line 4,233: | Line 5,281: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | + | ||
| − | Extraction-yield is down 5- to 10- fold compared to SDS (calculated from SDS-lysis results with 10 | + | Extraction-yield is down 5- to 10- fold compared to SDS (calculated from earlier SDS-lysis results with 10<sup>9</sup> (not 2*10<sup>9</sup>) |
| + | <img src="https://static.igem.org/mediawiki/2017/9/99/T--Munich--pic--lysis_heatvssds.png" style="max-width:60%"> | ||
</li> | </li> | ||
| + | </ul> | ||
</ul> | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
| Line 4,263: | Line 5,313: | ||
</ul> | </ul> | ||
| + | <div class="date">Thursday 14th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RPA stability test and Reaction Temperature Screening on paper </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Preparation of stability assay of RPA on paper. Lyophilisation and storage. | ||
| + | </li> | ||
| + | <li> | ||
| + | RPA on paper according to protocol | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, Ligation of DNA fragments, Gibbson-Assembly and E and chem Trafo</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | New digestion of unamplified N-Terminal gBlock and psB1C3 with SpeI-HF and EcoRI-Hf <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Dephosphorylation with Antarctic Phosphatase | ||
| + | </li> | ||
| + | <li> | ||
| + | Ligation of digested sequences overnight with T4 DNA Ligase | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson-Assembly of psB1C3with N-Term fragment | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation of chem and e. competent Turbo cells | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
<div class="date">Friday 15th</div> | <div class="date">Friday 15th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RPA stability test and Reaction Temperature Screening on paper </b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Stability of RPA on paper was tested using His6-TEV plasmid after 0, 2 and 24 hours post-lyophilisation. Reaction temperature was tested at 37 °C | ||
| + | as suggested by TwistDx and room temperature. Reaction was positively controlled using non-lyophilised reaction mixture. | ||
| + | </li> | ||
| + | <li> | ||
| + | RPA according to protocol | ||
| + | </li> | ||
| + | <li> | ||
| + | PCR cleanup according to protocol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Lyophilisation of RPA on paper worked.</li> | ||
| + | <li>Directly after lyophilisation, the reaction | ||
| + | efficiency seems to be similar to fresh RPA reaction mixture.</li> | ||
| + | <li>Lyophilisation of RPA on paper worked. However, activity loss is observed already after 2 hours and after 24 hours, | ||
| + | almost no activity is observable anymore. </li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/12/T--Munich--Results_20170915_RPA_Stability_1.png"> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Picked 32 colonies from plates of 14.09.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Screened with Colony PCR | ||
| + | </li> | ||
| + | <li> | ||
| + | Gel 200 V 2 % Agarose 30 minutes | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/5/54/T--Munich--Results_20170915_Max_Gel.png"> | ||
| + | <ul><li>No positive colononies in the Col PCR.</li></ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,288: | Line 5,475: | ||
</li> | </li> | ||
</ul> | </ul> | ||
| + | <div class="date">Monday 18th</div> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| − | <b>RPA stability test | + | <b>RPA stability test on paper </b> |
</li> | </li> | ||
| Line 4,305: | Line 5,493: | ||
<ul> | <ul> | ||
<li> | <li> | ||
| − | + | Preparation of temperature and air-sensitivity assay of RPA on paper. | |
| − | + | ||
</li> | </li> | ||
<li> | <li> | ||
| − | RPA according to protocol | + | RPA on paper according to protocol. |
</li> | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Lyophilisation of RPA on paper.</li> | ||
| + | <li>Storage with and without Parafilm enclosure at 4 °C or room temperature.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and Restriction digestion and Col-PCR and e and chem comp Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
<li> | <li> | ||
| − | + | New PCR of backbone with psB1A3-fw and psB1A3-rev. Sample was subsequently DpnI digested and purified <!--All--> | |
| + | </li> | ||
| + | <li> | ||
| + | Col-Pcr of 16 non green colonies from plates from thursday 14.9.17 | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation of cells with ON Ligation from Friday 15.9.17 into E.coli DH5 alpha and E.coli Turbo | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <div class="date">Tuesday 19th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNA extraction from 5ml overnight culture of B. subtilis </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Phenol-chlorophorm extraction RNA</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | used heat lysis at 90 °C for 15 mins | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Coupling <i>In-Vitro Transcription</i> to RPA </b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. | ||
| + | The construct as template used was His6-TEV plasmid. | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | RPA-Tx according to protocol. | ||
| + | </li> | ||
| + | <li> | ||
| + | Phenol-Chloroform-Extraction according to protocol. | ||
</li> | </li> | ||
</ul> | </ul> | ||
| Line 4,318: | Line 5,588: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | + | <ul> | |
| − | + | <li>In-Vitro Transcription could not be shown.</li> | |
| − | + | <li>RNA was detected in the samples in which no T7 RNA Polymerase was added while there are no | |
| − | + | bands in the samples that had T7 RNA Polymerase in them. Though unlikely, a labelling error might have occured. </li> | |
| − | + | </ul> | |
| − | + | <img src="https://static.igem.org/mediawiki/2017/e/e6/T--Munich--Results_20170919_rna_gelly.png"> | |
</li> | </li> | ||
</ul> | </ul> | ||
| − | + | <ul class="myEntry other"> | |
| − | + | <li class="myTitel"> | |
| − | + | <b>RPA stability test on paper </b> | |
| − | + | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Stability of RPA on paper was tested using His6-TEV plasmid after 24 hours post-lyophilisation. Storage conditions were altered. | ||
| + | Storage at room temperature and at 4 °C were tested. Also, duplicates were done at these temperatures, one sample being always enclosed | ||
| + | with Parafilm to avoid air exposure. | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | RPA on Paper according to protocol | ||
| + | </li> | ||
| + | <li> | ||
| + | PCR cleanup according to protocol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>RPA activity clearly showed that storage temperature did not have a significant effect on the integrity of the RPA reaction mixture.</li> | ||
| + | <li>Exposure to air, however, was identified as the bottleneck for RPA stability. The RPA samples that were protected from exposure to air showed significantly higher</li> | ||
| + | </ul> | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/2/21/T--Munich--PagePictures_RPA_gel2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout and Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Gibbson-Assembly and Ligation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Rob <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Gel of Col-PCR from 18.09.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Clean-up from backbone PCR and digest from 18.09.17 followed by Gibbsonassembly with N-terminal insert. | ||
| + | </li> | ||
| + | <li> | ||
| + | Started the Recloning of the Biobricks by PCR with phosphorylated primers and T4 Ligation and transformed into Turbo chem competent cells. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/43/T--Munich--Results_20170919_Max_Gel.png"> | ||
| + | <ul><li>Sample 1-3 and 2-1 had the correct length, and therefore precultures of these colonies were started overnight.</li></ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>IC3a invitro transcription</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">In vitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Results_20170919_ICa3_invitro_transcription_scheme.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | See results of DNaseI digestion and PCE from 20.09.17. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,346: | Line 5,704: | ||
Notes: | Notes: | ||
<ul> | <ul> | ||
| − | 2x SDS-Heat lysis, 2x 90 °C | + | 2x SDS-Heat lysis (protocol), 2x heat-only at 90 °C for 20 min |
</ul> | </ul> | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | SDS-lysis works a lot worse for gram+ bacteria, Heat-only now better/same (more incubation time...) | + | SDS-lysis works a lot worse for gram+ bacteria, Heat-only now better/same than SDS-lysis (more incubation time...) |
| − | + | A260/280 ratio relatively bad, probably a lot of cell-wall impurities | |
| − | + | <img src="https://static.igem.org/mediawiki/2017/e/ee/T--Munich--pic--lysis_heatvssds_grampos.png" style="max-width:60%"> | |
</li> | </li> | ||
</ul> | </ul> | ||
| − | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Heat lysis and isothermal PCR: temperature dependence</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RPA-pcr</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian, Sven | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Diluted E. coli (DH5a-pSB1C3-Tev-008-His) suspension to 10<sup>6</sup> cells / ml and heat-lysed in 50 uL aliquots | ||
| + | </li> | ||
| + | <li> | ||
| + | Lysis-temperature ranged from 65 °C to 90 °C in 5 °C steps | ||
| + | </li> | ||
| + | <li> | ||
| + | Added 2 uL of lysed sample to 10 uL RPA-solution and TEV-protease primers | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Heat differences in this range and this cell concentration have only minor effect. Best lysis seems to be around 80 °C, which is consistens with literature. | ||
| + | <img style="margin-bottom: 15px" src="https://static.igem.org/mediawiki/2017/thumb/6/6e/T--Munich--pic--lysis_pcr_deltatemp.png/765px-T--Munich--pic--lysis_pcr_deltatemp.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="date">Wednesday 20th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Gel analysis of B.subtilis RNA samples </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Denaturating Urea PAGE</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | 8% gel | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | B. subtilis-PCE 190 ng/ul, B.subtilis heat/lysis-378 ng/ul | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Miniprep with Qiagen Kit</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Minipreped overnight cultures from 1-3 and 2-1. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Send samples for sequencing with VF and VR. | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | N-Terminal sample 1-3 was positive. <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col PCR and preparation of e comp. cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Colony PCR of 36 colonies from each of the 3 constructs His-Sumo-Lwa (Lwa(l)), Lwa(lwa(k) and His-Sumo-Lbu each in psB1C3. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Precultures of 4 colonies from Lwa(l) and Lwa(k) | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/b1/T--Munich--Results_20170920_Max_Gel.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>DNAse I digest and Phenol-Chloroform-Precipitation</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Restriction digest,Phenol-chlorophorm purification of RNA</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f8/T--Munich--Results_20170815_Digestion_PCE_scheme.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>6 M UREA-PAGE of invitro transcribed and pooled IC3a.</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f8/T--Munich--Results_20170815_Digestion_PCE_scheme.png"> | ||
| + | <ul> | ||
| + | <li>Quantitative gel analysis calculated a concentration of 35 µM.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Thurdsay 21st</div> | <div class="date">Thurdsay 21st</div> | ||
| − | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
| − | <div class="date"> | + | <li class="myTitel"> |
| − | + | <b>Intein-Extein-Readout</b> <!----> | |
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Digestion of 1 µg of each backbone (psb1c3_N-term_uni and psb4A5_C-Term_uni with BBS1-HF to open them for Gibbson-Assembly <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson-Assembly of Col_PCR fragments from 03.08.2017 | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li> | ||
| + | 1:3 backbone to insert concentration | ||
| + | </li> | ||
| + | <li> | ||
| + | 30 minutes at 50 °C | ||
| + | </li> | ||
| + | </ul> | ||
| + | <li> | ||
| + | Transformation of E.coli Turbo e and chem competent cells according to protocol | ||
| + | </li> | ||
| + | <li> | ||
| + | Preculture for Cryostock from psB1C3_Split_uni_N_term 1-3 | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR and Sample Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Send samples for sequencing <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Col_PCR of 4 samples from Lbu plate (all negative) | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <div class="date">Wednesday 20th</div> | ||
| + | <ul class="myEntry cs13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNase Alert Experiment with Bacillus subtilis using varying concentrations of target RNA </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Pippeting scheme: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--pic--platereader_pipScheme_20_09_bazillus.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="date">Thursday 21th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> B. subtilis RNA extraction using lysozyme </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Phenol-chlorophorm RNA extraction</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Monday 25th</div> | <div class="date">Monday 25th</div> | ||
| − | + | <ul class="myEntry other"> | |
| + | <li class="myTitel"> | ||
| + | <b> crRNA in vitro transcription (for B.subtilis, Norovirus. Q5 beta, Hepatitis) | ||
| + | </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">In vitro Transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, PCR Cleanup Kit, Gibbson Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Redigest of 1 µg of each backbone with BBS1 and dephoshorilation according to protocol | ||
| + | <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Cleanup with cleanup kit | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson assembly with 100 µg backbone and 3:1 excess of insert ( calculated based on length ratio) Inserts 2 and 3 from PCR of 05.08.17 | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation into E.coli Turbo e. comp | ||
| + | </li> | ||
| + | <li> | ||
| + | COlPCR of plates from Thursday. 21.9.17=> 36 samples | ||
| + | </li> | ||
| + | <li> | ||
| + | Gel at 1 % 45 mins 150 V | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | |||
| + | |||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_LBU <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gel at 1 % 45 mins 150 V | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | All negative. | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Tuesday 26th</div> | <div class="date">Tuesday 26th</div> | ||
| − | + | <ul class="myEntry other"> | |
| + | <li class="myTitel"> | ||
| + | <b> crRNA quantification, Urea gel run </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Denaturating Urea Page</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Erika | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | 2 samples each from B. subtilis, Norovirus, Q5 beta and HCV(first diluted 1:5 and second undiluted) | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, Agarosegel</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max, Milica <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | ColPCR of 12 colonies from N-term plate from 25.9.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gel at 1 % 30 mins 150 V | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | Samples 1-8 and 2-1 of the N-Terminus looked good => preculture for sequencing. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_Lwa_kurz <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gel at 1 % 30 mins 150 V | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | All negative. | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Wednesday 27th</div> | <div class="date">Wednesday 27th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Minipreparation and Sample Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Miniprep of psB1C3_Split_N_term_bGal 1-8 and 2-1 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Send Minipreps for sequencing with primer VF and VR | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | Both sequences positive. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,392: | Line 6,185: | ||
</ul> | </ul> | ||
<div class="date">Thursday 28th</div> | <div class="date">Thursday 28th</div> | ||
| − | + | <ul class="myEntry other"> | |
| + | <li class="myTitel"> | ||
| + | <b> Agarose and Urea gel run with RNA </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">PAGE, Urea PAGE</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Erika | ||
| + | </li> | ||
| + | |||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Agarose Gel didn’t work – The samples seems to be degraded in agarose gel , may be due to contamination of the gel chamber and other materials with Rnase. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, ON culture</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | ColPCR from samples of 22.09.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | 32x Lwa short | ||
| + | </li> | ||
| + | <li> | ||
| + | Overnight cultures from colonies 1-8 and 2-3 started | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/0e/T--Munich--Results_20170928_Max_Gel.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RNA/DNA hybrid dimerization with Cy5 DNA-oligo C3a</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c0/T--Munich--Results_20170928_RNA_DNA_hybridization_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Native PAGE 20%, 12.5 mM MgCl2</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/7/77/T--Munich--Results_20170928_RNA_DNA_hybridization_gel.png"> | ||
| + | <ul> | ||
| + | <li>C3a too high concentrated (Red bands). 5 µM instead of 500 nM. Better results with band shift see 29-09-2017.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Screen of LBU activity on RNA/DNA dimer</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNAse activity protocol</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c9/T--Munich--Results_20170928_Screening_Lbu_DNA_RNA_dimer_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Native PAGE 20%, 12.5 mM MgCl2</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d6/T--Munich--Results_20170928_Screening_Lbu_DNA_RNA_dimer_gel.png"> | ||
| + | <ul> | ||
| + | <li>No visible shift of activator DNA oligo with addition of RNA under the use of Cy5 modified DNA activator oligo. Results are bad because of too high C3A DNA oligo concentration. (C3a = red bands).</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Friday 29th</div> | <div class="date">Friday 29th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNaseAlert experiment with extracted RNA targets from E.coli and in vitro transcribed RNAs. </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Erika | ||
| + | </li> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Pipetting scheme: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/e5/T--Munich--pic--platereader_pipScheme_29_09_ecoli.png"> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | The results for E.coli look good. Excel table saved in lrz as: 2017_09_29_rnase alert_ecolitarget_invitrotarget.xlsx | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prepatation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Mini prep of 1-8 and 2-3. | ||
| + | </li> | ||
| + | <li> | ||
| + | Lwa short send for sequencing with VF and VR <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | Lwa long positive. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>DNA and RNA hybrid of 3C-A and IC3-A</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c0/T--Munich--Results_20170929_DNA_RNA_hybridization_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Native PAGE 20%, 12.5 mM MgCl2</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/b4/T--Munich--Results_20170929_DNA_RNA_hybridization_gel.png"> | ||
| + | <ul> | ||
| + | <li>Visible small up shift of RNA/DNA dimer versus samples, which only contain the DNA oligo by itself. (DNA oligo C3a marked with Cy5 visible as red bands.)</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,489: | Line 6,465: | ||
<div class="month" id="October" > | <div class="month" id="October" > | ||
<div class="date fistOfMonth">Monday 2nd</div> | <div class="date fistOfMonth">Monday 2nd</div> | ||
| − | + | <ul class="myEntry other"> | |
| + | <li class="myTitel"> | ||
| + | <b> Phenol chloroform purification of crRNAs of B. subtilis, NoroV, HCV and Q5 beta | ||
| + | </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Phenol-chloroform extraction RNA</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">In vitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/9/9a/T--Munich--Results_20171002_live_invitro_trasncription_scheme.png"> | ||
| + | <ul> | ||
| + | <li>Forgot to add C3ds with single strand overhang, which is needed for activation of invitro transcription after binding of DNA oligo C3a (activator of C3ds).</li> | ||
| + | </ul> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>Plate reader data showed no activity for all samples, as expected without DNA template to transcribe.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Tuesday 3rd</div> | <div class="date">Tuesday 3rd</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> various Rnase alert experiments </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | with Noro Virus (10 ul total volume used for the experiment) </br> | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | with Q5 beta (10 ul total volume used for the experiment) | ||
| + | </li> | ||
| + | <li> | ||
| + | with Bacillus ( 10 ul total volume used for the experiment) and | ||
| + | </li> | ||
| + | <li> | ||
| + | with HCV and E.coli RNA (FINA method) samples (10 ul total volume used for the experiment) | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting Scheme Norovirus and Q5 beta: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/a2/T--Munich--pic--platereader_pipScheme_03_10_norovirus_q5beta.png"> | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting Scheme JVC and B. subtilis: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/e8/T--Munich--pic--platereader_pipScheme_03_10_hvc_bacillus.png"> | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting Scheme E.coli RNA (FINA method): </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/4e/T--Munich--pic--platereader_pipScheme_03_10_ecoli.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Bacillus, Noro virus and HCV seeem to work. However Q5 beta needs to be repeated and also HCV could be repeated! | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> FINA extraction of RNA from E. coli with and without filters </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">FINA RNA extraction</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Jorge | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Works, Target-sequence with PCR detected. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Live Invitro Transcription test with LBU and Inhibitor-RNA/Activator-DNA dimer with different T7 concentration</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">In vitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/e/ed/T--Munich--Results_20171003_live_invitro_trasncription_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/2a/T--Munich--Results_20171003_live_invitro_trasncription_results.png"> | ||
| + | <ul> | ||
| + | <li>No Invitro transcription of RNA at any T7-Polymerase concentration. LBU does either not set activator DNA free or does not work at all. Same result as on the gel.</li> | ||
| + | <li>RNAse H shows low level fluorescence over the whole experiment, which can not be related to an invitro transcription.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Wednesday 4th</div> | <div class="date">Wednesday 4th</div> | ||
| − | + | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Bacillus RNA extraction </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Erika, Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | |||
| + | Resuspension Buffer (20 ml): | ||
| + | <li> | ||
| + | Sucrose (0.3 M) -> 2.1 g | ||
| + | </li> | ||
| + | <li> | ||
| + | Sodium Acetate (10 mM) -> 16.4 mg | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | Adjust pH to 4.2 | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | Stored at 4°C | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Send Lwa short colony 2-3 for sequencing with pseqLwa primer <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | Full read with no mutations. | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Thursday 5th</div> | <div class="date">Thursday 5th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNaseAlert </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Rob, Erika | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | RNaseAlert reaction mix ( in glass fiber filter paper) lyophilisation. Introduction and first trial | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Repeated Gibbson-Assembly for Lbu with 4:1 ratio<!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation into E.coli Turbo | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | Empty plates => need to repeat PCR. | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,510: | Line 6,720: | ||
<li class="myObservations"> | <li class="myObservations"> | ||
</li> | </li> | ||
| − | <li class="myResults"> | + | <li class="myResults"> |
| − | + | ||
| − | + | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Friday 6th</div> | <div class="date">Friday 6th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNA extraction from Bacillus overnight culture </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">Phenol-chlorophorm extraction</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Erika | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | following SIGMA Genosys Protocol from Step 1 to 7. Then, followed Lab´s protocol steps. | ||
| + | </li> | ||
| + | <li> | ||
| + | RNA sample stored at -80°C and needs to be quantified. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,556: | Line 6,786: | ||
</ul> | </ul> | ||
<div class="date">Saturday 7th</div> | <div class="date">Saturday 7th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>SybrGreen II sensitivity test</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/4/49/T--Munich--Results_20171007_Sybr_Green_activity_test_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c5/T--Munich--Results_20171007_Sybr_Green_activity_test_results.png"> | ||
| + | <ul> | ||
| + | <li>1:10.000 seems to be the right dilution of SybrGreen II dye for a 15 µl Sample.</li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,591: | Line 6,846: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Sunday 8th</div> | <div class="date">Sunday 8th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>IC3A invitro transcription</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Invitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/8/83/T--Munich--Results_20171008_Invitrio_trasncriptipon_IC3a_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | See results of Digestion and PCE from 09.10.17. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>T7 polymerase sensitivity test</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Invitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/9/99/T--Munich--Results_20171008_T7_activity_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/28/T--Munich--Results_20171008_T7_activity_results.png"> | ||
| + | <ul> | ||
| + | <li>Higher concentrations of T7-polymerase causes the maximal concentration of RNA to be reached faster.</li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,636: | Line 6,934: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | + | <img src="https://static.igem.org/mediawiki/2017/c/c3/T--Munich--Results_20171008_IVTX_AuNP_linker_1-10.png"> | |
| + | <img src="https://static.igem.org/mediawiki/2017/3/36/T--Munich--Results_20171008_IVTX_AuNP_linker_15.png"> | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Monday 9th</div> | <div class="date">Monday 9th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Quantification of viral RNAs and Bacillus RNA extracted from last week </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNA urea gel</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Lbu-Recloning with Gibbson-Assembly</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR,Gibbson and Trafo in chem and e comp</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | PCR for Gibbson with Biobrick fwd and rev primer. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Lbu Gibbson Assembly with 3x excess of insert. | ||
| + | </li> | ||
| + | <li> | ||
| + | Trafo into E. coli chem and e comp. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | No colonies | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>DNAse I digest and PCE of 4 pooled invitro transcriptions of IC3a</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Restriction digest,Phenol-chlorophorm purification of RNA</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f8/T--Munich--Results_20170815_Digestion_PCE_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>6 M UREA-PAGE 15% of IC3a RNA</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/48/T--Munich--Results_20171009_Digestion_PCE_gel.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/7/75/T--Munich--Results_20171009_Digestion_PCE_results.png"> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="date">Tuesday 10th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR and Gibbson Assembly</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | PCR of Lbu and psB1C3 as backbone <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson Assembly with 5:1 excess of insert | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Heat lysis and isothermal PCR: cell concentration dependence s</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RPA-pcr</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Julian, Sven | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Diluted E. coli (DH5a-pSB1C3-Tev-008-His) suspension in a range of 10<sup>6</sup> to 10<sup>2</sup> cells / ml and heat-lysed in 50 uL aliquots | ||
| + | </li> | ||
| + | <li> | ||
| + | Added 2 uL of lysed sample to 10 uL RPA-solution and TEV-protease primers | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Bands visible down to a dillution of 5*10<sup>4</sup> or 10<sup>4</sup> cells/ml, which corresponds to an average of 100 or 20 cells' DNA per PCR | ||
| + | <img style="margin-bottom: 15px" src="https://static.igem.org/mediawiki/2017/e/e0/T--Munich--pic--lysis_pcr_deltacells.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
<div class="date">Wednesday 11th</div> | <div class="date">Wednesday 11th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Plate reader experiments </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Jorge | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | with Noro virus samples ( worked!) | ||
| + | </li> | ||
| + | <li> | ||
| + | with Bacillus ( seems to have Rnase contamination) | ||
| + | </li> | ||
| + | <li> | ||
| + | with the paper strips (glass fiber filter paper) 1.8 ul reaction mix pipetted with different amounts of invitro transcribed RNAs. | ||
| + | </li> | ||
| + | <li> | ||
| + | with E.coli RNA samples( heat lysed)- ( seems to have RNase contamination) | ||
| + | </li> | ||
| + | |||
| + | <li> | ||
| + | Pipetting Scheme 1: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/ea/T--Munich--pic--platereader_pipScheme_10_10_div1.png"> | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting Scheme 2: </br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--pic--platereader_pipScheme_10_10_div2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Coupling <i>In-Vitro Transcription</i> to RPA in Bulk and on Paper</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. | ||
| + | The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | RPA-Tx and RPA-Tx on Paper according to protocol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Preparation: <br> | ||
| + | Solubilisation of G-Block to reach 10 ng/ul concentration. <br> | ||
| + | PCR reaction using VF and VR as primers resulted in 50 ul of a 24 ng/ul solution. <br> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibson Assembly: <br> | ||
| + | PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 54 ng/ul. | ||
| + | Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. | ||
| + | </li> | ||
| + | <li> | ||
| + | Restriction Cloning:<br> | ||
| + | Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. <br> | ||
| + | Subsequent dephosphorylation of backbone using Antartic Phosphatase. <br> | ||
| + | T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on | ||
| + | chloramphenicol agar plates. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | The investigation of the agar plates the next day using UV-light revealed that the only plasmid that was taken up by the Turbo cells | ||
| + | was the initial pSB1C3-GFP plasmid. No positive inserts could be identified. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning </b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of chem comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of Turbo cells with Gibbson of 10.10.<!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Dye Test for live imaging of invitro transcription (1st row Controls (with SybrGold partially), 2nd row SybrGold dilution series, 3rd row SybrGreen I dilution series, 4th row EvaGreen dilution series)</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">invitro transcription</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/7/7e/T--Munich--Results_20171011_live_invitro_transcription_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>6 M UREA-PAGE 15% of IC3a RNA</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/b0/T--Munich--Results_20171011_live_invitro_transcription_results.png"> | ||
| + | <ul> | ||
| + | <li>Mistake made: Added T7 Polymerase to Control 2. Was left out in Graph.</li> | ||
| + | <li>Only Eva Green seems to not influence the invitro transcription, because it was especially designed for live imaging with ongoing transcription reactions.</li> | ||
| + | </ul> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
| Line 4,700: | Line 7,295: | ||
<div class="date">Thursday 12th</div> | <div class="date">Thursday 12th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Plate reader experiment with paper strips blocked ovenight with BSA </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. | ||
| + | </li> | ||
| + | <li> | ||
| + | The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting scheme: <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/30/T--Munich--pic--platereader_pipScheme_12_10_paper.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | Seems to work! Needs to be repeated for the confirmation of results. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>UREA-PAGE of Coupling <i>In-Vitro Transcription</i> to RPA in Bulk and on Paper</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. | ||
| + | The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. | ||
| + | |||
| + | </li> | ||
| + | <li> | ||
| + | RPA-Tx and RPA-Tx on Paper according to protocol. | ||
| + | </li> | ||
| + | <li> | ||
| + | UREA-PAGE Gel of Samples extracted via Phenol-Choloform-Extraction; both according to protocol. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>T7 RNA-Polymerase was, by accident, also added to the negative control of the RPA-Tx reaction on Paper. </li> | ||
| + | <li>First, one can see that the gel was overloaded. Also, it seems like there is additional bands at the expected size of around 150 bp.</li> | ||
| + | <li>Questionable is that this seems also to be the case for the negative Control of the RPA-Tx reaction in Bulk. </li> | ||
| + | <li>The experiment on paper, however, looks quite promising since it seems like there are distinct bands at the expected approximate size of the RNA transcript. Cas13a cleavage assay will provide evidence if these bands actually consist of the desired target RNA. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/ad/T--Munich--Results_20171012_Urea_PAGE_RNA_RPA.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/c3/T--Munich--Results_20171012_Urea_PAGE_RNA_RPATx_bulk.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of BL21 with psB4A5 C-Term and psB1C3 N-Term <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | No colonies for psB4A5 C-Term. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR for Biobrick Recloning</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Col PCR of 6x N-Term in psB1C3, 16x C-Term in psB1C3, 14x Lbu in psB1C3 <!--All--> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | All negative. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Eva Green Sensitivity Test with RNA background</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | |||
| + | <img src="https://static.igem.org/mediawiki/2017/2/2f/T--Munich--Results_20171012_Eva_sensitivity_scheme.png"> | ||
| + | |||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,724: | Line 7,468: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Friday 13th</div> | <div class="date">Friday 13th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Plate reader experiment with paper strips blocked ovenight with BSA </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Rob | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | |||
| + | |||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. | ||
| + | </li> | ||
| + | <li> | ||
| + | The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. (Results seem to work!) | ||
| + | </li> | ||
| + | </ul> | ||
| + | <li> | ||
| + | Pipetting scheme: <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/6b/T--Munich--pic--platereader_pipScheme_14_10_paper.png"> | ||
| + | </li> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5-Polymerase</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Repeated PCR of N and C-term part for Recloning <!--All--> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | |||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,750: | Line 7,545: | ||
</li> | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="date">Saturday 14th</div> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | DpnI digest of PCR from 31.10.17 <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Clean-up with PCR purification kit | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson-Assembly with 3:1 excess | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation into E.coli Turbo chem comp. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <div class="date">Sunday 15th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> RNA extraction of E. coli and Bacillus + plate reader experiment</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa, Jorge, Erika | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Notes: | ||
| + | <ul> | ||
| + | <li> | ||
| + | HCV assay and Q5beta in Fluorostar and Clariostar for 1 hour each (HCV assay worked!) | ||
| + | </li> | ||
| + | <li> | ||
| + | RNA urea gel for RNA extracted from E.coli and Bacillus | ||
| + | </li> | ||
| + | <li> | ||
| + | Plate reader experiment of RNA extracted from the E.coli in Fluorostar for 2 hours | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting scheme: <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f1/T--Munich--pic--platereader_pipScheme_15_10_hvc.png" | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Repeated Transformation of Bl21 star with psB4A5-Split-Intein-C-Term <!--All--> | ||
| + | </li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
| − | + | <!----> | |
| + | No colonies.=> Transformation in other expression strains | ||
</li> | </li> | ||
| − | </ul> | + | </ul> |
| + | |||
<div class="date">Monday 16th</div> | <div class="date">Monday 16th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. | ||
| + | </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Pipetting scheme: <br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/bc/T--Munich--pic--platereader_pipScheme_16_10_paper.png" > | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Transformed all available Bl21 strains with psB4A5-C-Term <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Strains were: | ||
| + | </li> | ||
| + | <li> | ||
| + | Bl21 pLYs; BL21 star; BL21 DE3, BL21 RIPL, Rosetta and E.coli Turbo as pos control | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | No colonies for any BL21 strain. The Turbo cells grew fine. => Expression seems to be toxic. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,787: | Line 7,720: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Tuesday 17th</div> | <div class="date">Tuesday 17th</div> | ||
| + | <ul class="myEntry cas13a"> | ||
| + | <li class="myTitel"> | ||
| + | <b> Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. | ||
| + | </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="#">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawa | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | <ul> | ||
| + | <li> | ||
| + | Pipetting Scheme B. subtilis:<br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--platereader_pipScheme_17_10_bacillus.png"> | ||
| + | </li> | ||
| + | <li> | ||
| + | Pipetting Scheme RPA paper:<br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Munich--pic--platereader_pipScheme_17_10_paper.png"> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Intein-Extein-Readout</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Midipreparation of psB4A5-C-Term with Macherey-Nagel Xtra Midi <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Preparation done according to protocol | ||
| + | </li> | ||
| + | <li> | ||
| + | Plasmid was intended for In vitro Expression | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | In vitro expression was not done, due to lack of time. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR, Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | PCR of all GFP degradation tag library variants with Biobrick sequence binding primers, for transfer from psB1A3 to psB1C3. <!--All--> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibbson-Assembly and Transformation according to protocol | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
| + | |||
<ul class="myEntry"> | <ul class="myEntry"> | ||
| Line 4,818: | Line 7,840: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Wednesday 18th</div> | <div class="date">Wednesday 18th</div> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Overnight culture</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Picked 2 green colonies from each plate for overnight culture <!--All--> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | |||
| + | |||
| + | </ul> | ||
| + | |||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,848: | Line 7,893: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Thursday 19th</div> | <div class="date">Thursday 19th</div> | ||
| − | + | <ul class="myEntry other"> | |
| + | <li class="myTitel"> | ||
| + | <b>G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Preparation: <br> | ||
| + | PCR reaction of G-Block using VF and VR as primers resulted in 50 ul of a 27 ng/ul solution. <br> | ||
| + | </li> | ||
| + | <li> | ||
| + | Gibson Assembly: <br> | ||
| + | PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 52 ng/ul. | ||
| + | Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. | ||
| + | </li> | ||
| + | <li> | ||
| + | Restriction Cloning:<br> | ||
| + | Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. <br> | ||
| + | Simultanous dephosphorylation of backbone using Antartic Phosphatase. <br> | ||
| + | T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. | ||
| + | </li> | ||
| + | <li> | ||
| + | Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on | ||
| + | chloramphenicol agar plates. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. </li> | ||
| + | <li>Colony PCR was performed and showed inserts for several plasmids.</li> | ||
| + | <li>Overnight cultures were inocculated for sequencing.</li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/68/T--Munich--Results_20171019_Colony_PCR_RNA_amp.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/01/T--Munich--Results_20171019_RNA_M_AMP.png"> | ||
| + | </li> | ||
| + | |||
| + | </ul> | ||
| + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
| + | <li class="myTitel"> | ||
| + | <b>Biobrick Recloning</b> <!----> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Max <!-- --> | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Send samples from degradation tag library for sequencing <!--All--> | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <!----> | ||
| + | pdt2-E 2 was positive | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
<div class="date">Friday 20th</div> | <div class="date">Friday 20th</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>G-Block cloning of RNA-Amplification test plasmid</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Mini-prep of previously inocculated overnight cultures and sent to sequencing. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li>The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | |||
| + | <li class="myTitel"> | ||
| + | <b>In vitro transcription of crRNA Lbu, crRNA Lsh and target RNA at 37 ºC overnight</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">In vitro Transcription</a> | ||
| + | |||
| + | </li> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | See results of digestion and PEC from 21.10.17 | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,883: | Line 8,052: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | <h4>Results</h4> | + | </li> |
| − | + | </ul> | |
| + | <div class="date">Saturday 21st</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | |||
| + | <li class="myTitel"> | ||
| + | <b>DNAse I digest and PCE of the RNA crRNA from Lbu and Lsh</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Restriction digest,Phenol/Chlorophorm purification of RNA</a> | ||
| + | |||
| + | </li> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d2/T--Munich--Results_20171021_Lsh_gel.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/ea/T--Munich--Results_20171021_Lsh_gel2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | |||
| + | <li class="myTitel"> | ||
| + | <b>In vitro transcription of crRNA Lwa at 37 C overnight</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">In vitro Transcription</a> | ||
| + | |||
| + | </li> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Florian | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | See results of digestion and PEC from 23.10.17 | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Sunday 22nd</div> | <div class="date">Sunday 22nd</div> | ||
| + | <ul class="myEntry other"> | ||
| + | <li class="myTitel"> | ||
| + | <b>RPA on PDMS Chip</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">RPA on Paper</a> | ||
| + | |||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Sven | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <ul> | ||
| + | <li> | ||
| + | Since the PDMS chip was leaky, no real sample could be extracted from it. | ||
| + | </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <ul> | ||
| + | <li> Positive Controls as well as colony-PCR using RPA showed clear bands on a 2%-agarose gel. The RPA performed on Chip, however, was not showing any bands though Nanodrop concentration showed 27 ng/ul in the sample. </li> | ||
| + | </ul> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/2f/T--Munich--Results_20171022_RPA_on_Chip.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,913: | Line 8,156: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | <h4>Results</h4> | + | </li> |
| − | + | </ul> | |
| + | <ul class="myEntry readout"> | ||
| + | |||
| + | <li class="myTitel"> | ||
| + | <b>DNAse I digest and PCE of the RNA crRNA from Lwa</b> | ||
| + | |||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">Restriction digest,Phenol/Chlorophorm purification of RNA</a> | ||
| + | |||
| + | </li> | ||
| + | |||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | |||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/9d/T--Munich--20171022_Lwa_gel.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls with Lsh Cas13a </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/31/T--Munich--Results_20171022_plate_reader_scheme.png" | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/10/T--Munich--Results_20171022_plate_reader_results.png"> | ||
| + | <ul> | ||
| + | <li>No activity of Lsh Cas13a visible.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative control with TDPs from team TU Delft</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/66/T--Munich--Results_20171022_plate_reader_scheme2.png" | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/e/e8/T--Munich--Results_20171022_plate_reader_results2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with different concentrations of RNA extracted with chemical lysis and phenol chloroform purification from the E.coli culture</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/cb/T--Munich--Results_20171022_plate_reader_scheme3.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/94/T--Munich--Results_20171022_plate_reader_results3.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Repeat of plate reader experiment with Lsh Cas13a with Elution 1 and Elution 4,2</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/de/T--Munich--Results_20171022_plate_reader_scheme4.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/1a/T--Munich--Results_20171022_plate_reader_results4.png"> | ||
| + | <ul> | ||
| + | <li>No activity of Lsh Cas13a visible</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Cross target plate reader experiment using crRNA E.coli with different target RNAs from B.subtilis and Noro virus</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/5/5f/T--Munich--Results_20171022_plate_reader_scheme5.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/ff/T--Munich--Results_20171022_plate_reader_results5.png"> | ||
| + | <ul> | ||
| + | <li>It didn't work</li> | ||
| + | </ul> | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Monday 23rd</div> | <div class="date">Monday 23rd</div> | ||
| − | + | <ul class="myEntry readout"> | |
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with dried and not dried Cas13a and TDPs with different target concentrations and positive and negative controls</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/b5/T--Munich--Results_20171023_plate_reader_scheme.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f2/T--Munich--Results_20171023_plate_reader_results.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with with Cas13a Lbu and different concentrations of RPA samples </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d8/T--Munich--Results_20171023_plate_reader_scheme2.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/40/T--Munich--Results_20171023_plate_reader_results2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with old Lwa Cas13a elution</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d3/T--Munich--Results_20171023_plate_reader_scheme3.png"> | ||
| + | <ul> | ||
| + | <li>An elution from July, stored at 4 ºC of Lwa Cas13a was used</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/7/7e/T--Munich--Results_20171023_plate_reader_results3.png"> | ||
| + | <ul> | ||
| + | <li>It didn't work</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<div class="date">Tuesday 24th</div> | <div class="date">Tuesday 24th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with Lwa Cas13a, old elutions 2 and 3</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/1a/T--Munich--Results_20171024_plate_reader_scheme.png"> | ||
| + | <ul> | ||
| + | <li>An elution from July, stored at 4 ºC of Lwa Cas13a was used</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d5/T--Munich--Results_20171024_plate_reader_results.png"> | ||
| + | <ul> | ||
| + | <li>It didn't work</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with cross targets using crRNA form E.coli and different target RNAs form B. subtilis, Noro virus and E.coli</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/cf/T--Munich--Results_20171024_plate_reader_scheme2.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/06/T--Munich--Results_20171024_plate_reader_results2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with Spinach aptamer using Lbu Cas13a and different concentrations of in vitro targets</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/af/T--Munich--Results_20171024_plate_reader_scheme3.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--Results_20171024_plate_reader_results3.png"> | ||
| + | <ul> | ||
| + | <li>It didn't work</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,952: | Line 8,448: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Wednesday 25th</div> | <div class="date">Wednesday 25th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiments with lyophilized reaction mix with Cas13a Lbu with different concentrations of the in vitro target. </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/95/T--Munich--Results_20171025_plate_reader_scheme.png"> | ||
| + | <ul> | ||
| + | <li>Cas13a was added only immediately before starting the measurement.</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/22/T--Munich--Results_20171025_plate_reader_results.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with different concentration of heat lysed E.coli samples with Cas13a Lbu</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/97/T--Munich--Results_20171025_plate_reader_scheme2.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/b8/T--Munich--Results_20171025_plate_reader_results2.png"> | ||
| + | <ul> | ||
| + | <li>The activity of Cas13a is visible, but additional RNase activity was also seen. </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>General control plate reader experiments with Cas13a Lbu with everything, without crRNA, without target, without Cas13a, positive and negative control.</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/d/d8/T--Munich--Results_20171025_plate_reader_scheme3.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/2/2e/T--Munich--Results_20171025_plate_reader_results3.png"> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 4,986: | Line 8,543: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Thursday 26th</div> | <div class="date">Thursday 26th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with Noro virus crRNA, Lbu Cas13a and different RNA targets from E.coli, HCV and Noro virus.</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/9/99/T--Munich--Results_20171026_plate_reader_scheme.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/4/41/T--Munich--Results_20171026_plate_reader_results.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiment with Lwa Cas13a purified from the stored Lwa pellet using different concentrations of invitro transcribed RNAs </b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f2/T--Munich--Results_20171026_plate_reader_scheme2.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/7/77/T--Munich--Results_20171026_plate_reader_results2.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiments with the lyophilized reaction mix on paper using different concentrations of target RNAs</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/a/a2/T--Munich--Results_20171026_plate_reader_scheme3.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/04/T--Munich--Results_20171026_plate_reader_results3.png"> | ||
| + | <ul> | ||
| + | <li>It didn’t work since the lyophilisation was done overnight which was way too long for a small amount of 15 ul reaction mix </li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiments with the RNA aptamer Spinach with different concentrations of in vitro RNA samples</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/8/80/T--Munich--Results_20171026_plate_reader_scheme4.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/3/39/T--Munich--Results_20171026_plate_reader_results4.png"> | ||
| + | </li> | ||
| + | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
| Line 5,021: | Line 8,655: | ||
</li> | </li> | ||
<li class="myResults"> | <li class="myResults"> | ||
| − | |||
| − | |||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Friday 27th</div> | <div class="date">Friday 27th</div> | ||
| + | <ul class="myEntry readout"> | ||
| + | <li class="myTitel"> | ||
| + | <b>Plate reader experiments with Lwa ( elution 1 and 2 and wash 1 ) purified using Nickel NTA</b> | ||
| + | </li> | ||
| + | <li class="myProtocol"> | ||
| + | Protocol: <a href="/Team:Munich/Protocols">RNase activity</a> | ||
| + | </li> | ||
| + | <li class="myParticipants"> | ||
| + | Participants: Dawafuti | ||
| + | </li> | ||
| + | <li class="myObservations"> | ||
| + | Observations: | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/02/T--Munich--20171027_plate_reader_scheme.png"> | ||
| + | </li> | ||
| + | <li class="myResults"> | ||
| + | <h4>Results</h4> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/c/cf/T--Munich--Results_20171027_plate_reader_results.png"> | ||
| + | </li> | ||
| + | </ul> | ||
| − | |||
<div class="date">Tuesday 31st</div> | <div class="date">Tuesday 31st</div> | ||
| Line 5,036: | Line 8,686: | ||
<div class="three wide column" id="monthsList"> | <div class="three wide column" id="monthsList"> | ||
<ul> | <ul> | ||
| − | |||
<li><a href="#April">April</a></li> | <li><a href="#April">April</a></li> | ||
<li><a href="#May">May</a></li> | <li><a href="#May">May</a></li> | ||
Latest revision as of 11:45, 1 November 2017
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
