Difference between revisions of "Team:Munich/Testing"

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    <li class="myResults">
 
    <li class="myResults">
 
    Results
 
    Results
 +
    </li>
 +
    </ul>
 +
    <ul class="myEntry other">
 +
    <li class="myTitel">
 +
    <b>Lysis-tests with acidic phenol chlorophorm extraction </b>
 +
 +
    </li>
 +
    <li class="myProtocol">
 +
                        Protocol:  <a href="#">Alkaline lysis</a>
 +
    Protocol:  <a href="#">Phenol-Chlorophorm extraction</a>
 +
    Protocol:  <a href="#">Trizol Reagenz protocol</a>
 +
 +
    </li>
 +
    <li class="myParticipants">
 +
    Participants: Julian, Patrick
 +
 +
    </li>
 +
    <li class="myObservations">
 +
    Notes:
 +
    <ul>
 +
    <li>
 +
    Three lysis methods were used: Alkaline Lysis, heat lysis with SDS and Trizol-Lyse.
 +
    </li>
 +
    <li>
 +
    No vortexing for lysis (may break up cells and won't be available in our device), only room-temperature (apart form SDS as reference)
 +
    </li>
 +
    <li>
 +
    Cells had OD of 5.97 -> 4.78*10^9 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
 +
    </li>
 +
    <li>
 +
    Cells had OD of 5.97 -> 4.78*10^9 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 10^7 cells
 +
    </li>
 +
    </ul>
 +
    </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 -&gt; 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 -&gt;Ice, TROzol to -20! (100 miL at OD 1 -&gt; 10^7 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 &lt;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 10^7 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>-&gt; discard everything else, prot &amp; 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>-&gt; 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 -&gt; </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 -&gt; No RNA pellet or fog any more -&gt;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 -&gt; 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>-&gt; 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>-&gt; 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>-&gt;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">
 +
    <h4>Results</h4>
 +
                        No pellets seen during procedure, concentration too low for photometer -> repeat with more cells
 
    </li>
 
    </li>
 
    </ul>
 
    </ul>

Revision as of 17:46, 15 October 2017

Description

Design

Demonstrate

Measurement

Applied Design

Final Results

Achievements

Protocols

Notebook

Safety

Parts

Improved Part

Part Collection

InterLab

Model

Software

Collaboration

Detector

Quake Valve

Paperstrips

Sample Processing

Silver

Gold

Engagement

Collaborations

Team members

Sponsors

Attributions

Gallery

Labbook

According to all known laws of aviation, there is no way a bee should be able to fly. Its wings are too small to get its fat little body off the ground. The bee, of course, flies anyway because bees don't care what humans think is impossible. Yellow, black. Yellow, black. Yellow, black. Yellow, black.

Tuesday 21st
  • Cas13a
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Read-out
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 22nd
  • Cas13a123
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Cas13a41234
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 23rd
  • REad-out
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Target
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 24th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 27th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 29th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 30th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 31rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 3rd
  • Cas13a
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Read-out
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 4th
  • Cas13a123
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Cas13a41234
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 5th
  • REad-out
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • Target
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 6th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 7th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 10th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 11th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 13th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 14th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 17th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 20th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 21st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 24th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 25th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 27th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 1st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 2nd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 3rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 4th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 5th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 8th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 9th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 10th
  • Test of different lysis methods for total RNA extraction with Norgen kit
  • Protocol: Total RNA Purification with Norgen Kit
  • Participants: Julian, Milica, Jorge
  • Observations:
    • Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS.
    • Lysozyme digestion: According to protocol.
    • Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min.

  • Results

    See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17 for gel picture.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 11th
  • Test of different lysis methods for Phenol/Chloroform total RNA extraction.
  • Protocol: Phenol/Chloroform purification of RNA
  • Participants: Milica, Sven
  • Observations:
    • Three lysis method were used: sonification, lysozyme digestion and heat lysis in SDS.
    • Lysozyme digestion: according to the total RNA purification with Norgen Kit protocol.
    • Heat lysis: Sample was incubated in 2% SDS at 75 °C for 15 min.
    • The samples were incubated at -80 °C overnight and the purification finished the next day.

  • Results

    See results from "Phenol/Chloroform purification of RNA from in-vitro transcription" from 11.05.17.
  • Phenol/Chloroform purification of RNA from in-vitro transcription
  • Protocol: Phenol/Chloroform purification of RNA
  • Participants: Dawafuti, Sven
  • Observations
    • The samples were incubated at -80 °C overnight and the purification finished the next day.

  • Results

Friday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 15th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 16th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 17th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 22nd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 23rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 24th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 25th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 29th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 30th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 31st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 1st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 2nd
  • Total RNA extraction of E. coli W3110 with Norgen kit
  • Protocol: Total RNA Purification with Norgen Kit
  • Participants: Jorge
  • Observations:
    • Instead of using TE and lysozyme to lyse the cells, they were resuspenden in 2% SDS in TAE and incubated at 75 ºC for 15 min.
    • The 6 samples were stored at -80 ºC and labeled T RNA #1-6
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 5th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
  • RNA Adsorption Test on first 3D print
  • Participants: Jorge
  • Notes:
    • Total RNA sample 5 used, prepared on Friday 02/06 by Jorge (Concentration: 126 ng/µl)
    • Procedure:
      • Take 1 µl presample from stock ; add with 2 µl nf H2O and 3 µl 2x RNA LD
      • Put 5 µl of sample on device
      • Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD
      • Wait 15 minutes
      • Take 1 µl of sample immediately, add with 2 µl nf H2O and 3 µl 2x RNA LD
      • Store at -80 °C
      • What still needs to be done:
        • Cook samples at 95 °C for 10 minutes and put directly on ice afterwards (to prevent refolding)
        • Load on Gel and quantify
Tuesday 6th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 7th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 8th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 9th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 13th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
  • Lysis-tests with acidic phenol chlorophorm extraction
  • Protocol: Alkaline lysis Protocol: Phenol-Chlorophorm extraction Protocol: Trizol Reagenz protocol
  • Participants: Julian, Patrick
  • Notes:
    • Three lysis methods were used: Alkaline Lysis, heat lysis with SDS and Trizol-Lyse.
    • No vortexing for lysis (may break up cells and won't be available in our device), only room-temperature (apart form SDS as reference)
    • Cells had OD of 5.97 -> 4.78*10^9 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
    • Cells had OD of 5.97 -> 4.78*10^9 cells according to http://www.genomics.agilent.com/biocalculators/calcODBacterial.jsp?_requestid=1029158
      -> dilluted 1:100 (to V=10mL) and pelleted 209 miL to get to 10^7 cells
    • A
    B
    C
    D
    1
    		MethodSDSNaOHTRIzol
    2
    		prepare1 mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS -> TE-Buffer + SDS...
    3
    		----Lysis1x3x1x
    4
    		separate to tubes Spin 300xG, 5 min ->Ice, TROzol to -20! (100 miL at OD 1 -> 10^7 cells, Max. according to TRIzolPaper100 miL bactSusp100 miL bact-susp100 miL bact-suspension
    5
    		No washing step, bad for mRNA (TRIzolP.) 300 µl (0.45 ml) TES resuspend pellet
    6
    		12,5 uL Inhibitor (40k U/ml, 1 U/miL required)15 min, 75 °C lysis, vortexone probe after another
    7
    		(Addition of inhibitor); Transform total volume to gel tuberesuspend thorougly in 66 µl (100 miL) Solution1
    8
    		132 µl (200 miL) Sol2, INVERT 4x
    9
    		wait 0, 1 ,3 min
    10
    		add 99 µl (150 miL) Sol3, INVERT 4x
    11
    		Total volume: 297 µl (0.45 ml)
    12
    		--phenChloExtr
    13
    		measure pH, add acid...meassure Trizol PH, add acid if needed (pH <5)
    14
    		add 600 µl [same V (0.45 ml)] Phe:chloro.add 1 mL TRIzol to 10^7 cells
    15
    		5 min incubation
    16
    		add 0.2 mL CHLOROPHORM
    17
    		incub 2.5 (2-3) min, RT
    18
    		centri 12k g 15 min 4°C
    19
    		angle 45°, extract top phase carefully-> discard everything else, prot & DNA-> which Vol for the TRIzol aqueous phase?
    20
    		add 0.4 (0.375) ml Isopropanol, wait 10 min, RT
    21
    		centrifuge 10 min 12k , 4°C ->
    22
    		Aditional step, because RNA pellet was not entierly spinned down: Centrifugation (16000 rcf, 5 min, 4 °C)big-pellet assay in fridge, but 1. interphase was punctured/gel(DNA?) stuck to pipette 2. s.u.
    23
    		Aditional step, because suspected RNA fog was not entirely spinned down: remove upper and lower RNA fog phase
    24
    		Additional step: Centrifugation (16000 rcf, 5 min, 4 °C)
    25
    		No RNA pellet or RNA fog any more :(
    26
    		Addition of 400 µl isopropanol -> No RNA pellet or fog any more ->prob. removed too much
    27
    		discard supernatant -> RNA in gel-like pellet!-> gel hardly visible, nearly draged it out of tube (stuck to pipette...)
    28
    		resuspend in 0.75 ml 70 % (75% ideally) ethanol-> storeed RNA at -20°C is stable up to 1 year...
    29
    		vortex, centri 5 min 7,5k 4°
    30
    		discard supernatant, air dry for 5-10 min (do NOT let completely dry out)
    31
    		resuspend in RNAse free water 30 µl (20-50 miL) ->measurement...

  • Results

    No pellets seen during procedure, concentration too low for photometer -> repeat with more cells
Wednesday 14th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 15th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 16th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 20th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 21st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 22nd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 23rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 27th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 29th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 30th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 3rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 4th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 5th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 6th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 7th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 10th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 11th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 13th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 14th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 17th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 20th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 21st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 24th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 25th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 27th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 31th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 1st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 2nd
  • InterLab Study calibration
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • Patch length correction was on in the plate reader.
    • Calibration for the plate reader experiment done with LUDOX and Fluorescein.
    • We saw no difference between LUDOX and water when measuring at an OD of 600 nm.
    • For the fluorescein measurement, the best gain was 600 using 485 nm/520 nm.
    • Plasmids from the plate 7 (positive and negative control, and devices 1-6) were resuspended.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 3rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 4th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 7th
  • InterLab Study Day 1: Transformation
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • Each plasmids was transformed two times: with chemical transformation and with electroporation, for a total of 16 plasmids.

  • Results

    • Every plate except the ones for Device 1 had colonies.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 8th
  • InterLab Study Day 2: Colony transfer to liquid culture
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • Device 1 was again transformed, as no colonies grew on the plate.
    • Liquid cultures for the 7 plasmids whose plates showed colonies (all except Device 1) were made in duplicate.
    • The plates were stored at -4 °C.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 9th
  • InterLab Study Day 3: Plate reader experiments
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates.
    • The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set.
    • Patch length correction was on in the plate reader.
    • Device 1 was again transformed, as no colonies grew on the plate.

  • Results

    • Transformation of Device 1 was successful.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 10th
  • InterLab Study Day 4: Preparation of O/N cultures for plate reader experiments
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • O/N liquid cultures in duplicate were set up from all 8 Devices.
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 11th
  • InterLab Study Day 5: Plate reader calibration and experiments
  • Protocol: IGEM HQ InterLab Study Protocol
  • Participants: Dawafuti Erika
  • Observations:
    • Patch length correction was off in the plate reader.
    • Calibration for the plate reader experiment done with LUDOX and Fluorescein.
    • We saw the difference in measurements between LUDOX and water.
    • After measuring the OD600 of the O/N cultures, they were diluted with LB+Cm to an OD600 of 0.02, in duplicates.
    • The absorbance and fluorescence of the diluted cultures were measured at 0, 2, 4 and 6 hours after the cultures were set.

  • Results

  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 14th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 15th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 16th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 17th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 21st
  • FINA extraction for DNA with E. coli W3110 pre-purified gDNA
  • Protocol: FINA Extraction for DNA
  • Participants: Jorge
  • Observations:
    • The gDNA was pre-purified with the Phenol/Chloroform method.
    • The gDNA solution was diluted 1:100, 1:1000, 1:100000, 1:10e6 and 1:10e8. Each dilution was then used as a sample for the FINA extraction.
    • From each dilution, two extractions were performed. One as in protocol (1N-5N), the other without the NaOH washing step (1-5).
    • As negative control (KK) FINA extraction was performed as in protocol with nuclease free water as sample.
  • Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples and purified gDNA from 21.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge
  • Observations
    • For the membranes, no template was directly pipette. Instead, the membranes were put in the tube.
    • p-bGal_N_N was used as forward primer
    • p-bGal_N_C was used as reverse primer
    • Annealing was performed at 61 ºC
    • The amplification step was 90 s long
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • 1N-5N: 1:100-1:10e8 dilutions FINA extraction performed as in protocol.
    • 1-5: 1:100-1:10e8 dilutions FINA extraction without washing step.
    • 1K-5K: 1:100-1:10e8 dilutions.
    • KK: negative control for FINA extraction (nuclease free water as sample).
Tuesday 22nd
  • FINA extraction for DNA with E. coli W3110 cell culture
  • Protocol: FINA Extraction for DNA
  • Participants: Jorge
  • Observations:
    • The extraction was done when the culture had an OD600 of 1.2.
    • The cells were diluted 1:1, 1:100 and 1:1000 before the extraction.
    • Each dilution was used as sample twice: as in protocol (1N-3N and P1N-P3N) and washed with 300 ul lysis buffer from RNA extraction kit (Norgen, P/N 17200) (1G-3G and P1G-P3G).
    • Membranes 1N-3N and 1G-3G were put in 40 ul nuclease free water and incubated at RT for 10 min in order for the bounded DNA to go into solution.
  • Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 22.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge
  • Observations
    • The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted.
    • p-bGal_N_N was used as forward primer.
    • p-bGal_N_C was used as reverse primer.
    • Annealing was performed at 61 ºC.
    • The amplification step was 90 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • 1N-3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol, loaded in gel after elution (no PCR).
    • 1G-3G: 1:1-1:1000 dilutions FINA extraction performed with lysis buffer as washing step, loaded in gel after elution (no PCR).
    • P1N-P3N: 1:1-1:1000 dilutions FINA extraction performed as in protocol.
    • P1G-P3G: 1:1-1:1000 dilutions FINA extraction with lysis buffer as washing buffer.
    • KK: negative control for FINA extraction (nuclease free water as sample).
Wednesday 23rd
  • FINA extraction for DNA with E. coli W3110 cell culture
  • Protocol: FINA Extraction for DNA
  • Participants: Jorge, Benedikt
  • Observations:
    • The extraction was done when the culture had an OD600 of 2.33.
    • The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction.
    • Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution.
  • Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 23.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge, Benedikt
  • Observations
    • The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted.
    • p-bGal_N_N was used as forward primer.
    • p-bGal_N_C was used as reverse primer.
    • Annealing was performed at 61 ºC.
    • The amplification step was 90 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Afte the PCR, only 2,5,L and KK were loaded in the gel, as the other tubes had no liquid.
    • The gel was loaded before it was submerged in TAE-buffer.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • 2: 1:100 dilution FINA extraction performed as in protocol
    • L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer.
    • KK: negative control for FINA extraction (LB-medium as sample).
Thursday 24th
  • FINA extraction for DNA with E. coli W3110 cell culture (Repetition of the experiment from 23.08.17)
  • Protocol: FINA extraction for DNA
  • Participants: Jorge, Benedikt
  • Observations:
    • As there were problems with the gel from the 23th, we repeated the experiment.
    • The extraction was done when the culture had an OD600 of 2.78.
    • The cells were diluted 1:1, 1:100, 1:10000, 1:10e6 and 1:10e8 before the extraction.
    • Each dilution was used as a sample for the FINA extraction. In addition, three 1:1 dilutions were used as samples for FINA extraction: two according to protocol and the third with 300 ul lysis buffer as washing buffer. One of the 1:1 membranes was put in nuclease free water and incubated at RT for 20 min in order for the bounded DNA to go into solution.
  • Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge, Benedikt
  • Observations
    • The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted.
    • p-bGal_N_N was used as forward primer.
    • p-bGal_N_C was used as reverse primer.
    • Annealing was performed at 61 ºC.
    • The amplification step was 90 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • 1-5: 1:1-1:10e8 dilutions FINA extraction performed as in protocol
    • L: 1:1 dilution FINA extraction performed with 300 ul lysis buffer as washing buffer.
    • KK: negative control for FINA extraction (LB-medium as sample).
  • Total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit
  • Protocol: Total RNA Purification with Norgen Kit
  • Participants: Jorge, Benedikt
  • Observations:
    • 2 tubes stored at -80 ºC. Labelled gRNA #1, gRNA #2.

  • Results

  • Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
  • Protocol: Phenol/Chloroform purification of RNA
  • Participants: Jorge
  • Observations:
    • The incubation step at -80 ºC was done, O/N.
    • 2 tubes stored at -80 ºC. Labelled gRNA PEC #1, gRNA PEC #2.

  • Results

Friday 25th
  • Continuation of Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT from yesterday
  • Protocol: Phenol/Chloroform purification for RNA
  • Participants: Jorge
  • Observations:
    • See entry from 24.08.17
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 29th
  • FINA extraction for DNA with E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
  • Protocol: FINA extraction for DNA
  • Participants: Jorge
  • Observations:
    • The extraction was done when the culture had an OD600 of 0.88.
    • Na: Extraction as in protocol. Cl: Washed with 10 mM HCl instead of NaOH. H: incubated 10 min at 80 ºC. L: 10 min incubated in 1 mg/ml lysozyme. LH: 10 min incubated in 1 mg/ml lysozyme at 80 ºC.
  • Q5-PCR for beta-galactosidase from E. coli with FINA-DNA samples from 24.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge
  • Observations
    • The membranes from the FINA extraction were directly put into the PCR-mix. No template DNA was directly pipetted.
    • p-bGal_N_N was used as forward primer.
    • p-bGal_N_C was used as reverse primer.
    • Annealing was performed at 61 ºC.
    • The amplification step was 90 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    See Q5-PCR from 30.08.17
  • FINA extraction for RNA with purified total RNA (gRNA PEC #1)
  • Protocol: FINA extraction for RNA
  • Participants: Jorge
  • Observations:
  • First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT purified total RNA (Phenol/Chloroform extraction from 24.08.17) and FINA-RNA eluate
  • Protocol: First strand cDNA synthesis
  • Participants: Jorge
  • Observations:
    • p-bGal_N_C was used as primer.
    • Sample gRNA PEC #1 from 24.08.17 was diluted 1:4 and used as sample P.
    • The eluate from the FINA extraction for RNA from 29.08.17 was used as sample S.
    • For both reactions (S and P), 6 ul sample were pipetted.
Wednesday 30th
  • Q5-PCR for beta-galactosidase from E. coli with cDNA samples from 29.08.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge
  • Observations:
    • p-bGal_N_N was used as forward primer.
    • p-bGal_N_C was used as reverse primer.
    • Annealing was performed at 61 ºC.
    • The amplification step was 90 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • Na: FINA extraction performed as in protocol.
    • Cl: FINA extraction performed with 10 mM HCl instead of NaOH.
    • L: Cell solution incubated 10 min in 1 mg/ml lysozyme. No washing step in FINA
    • H: Cell solution incubated 10 min at 80 ºC. No washing step in FINA
    • LH: Cell solution incubated 10 min at 80 ºC in 1 mg/ml lysozyme. No washing step in FINA
    • K-: negative control FINA (LB-medium used as sample).
    • P: 1:4 dilution of gRNA PEC #1 after reverse transcription.
    • S: 1:4 dilution of gRNA PEC #1 after FINA extraction for RNA and reverse transcription.
    • KK: Negative control for the cDNA synthesis (water instead of MuLV-RT used)
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 31st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 1st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 4th
  • Total RNA extraction of E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with Norgen kit
  • Protocol: Total RNA Purification with Norgen Kit
  • Participants: Jorge, Dawafuti
  • Observations:
    • Stored at -80 ºC as TRNA Kit #1-4
  • Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
  • Protocol: Protocol
  • Participants: Jorge, Dawafuti
  • Observations
    • Stored at -80 ºC as TRNA PC #1-4.

  • Results

    See results Urea-SDS-PAGE from 05.09.17.
Tuesday 5th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 6th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 7th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 8th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 11th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 13th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 15th
  • Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
  • Protocol: Phenol/Chloroform purification of RNA
  • Participants: Jorge
  • Observations:
    • Two samples stored at -80 ºC labeled TRNA Lsh #1 15.09.17 and TRNA Lsh #2 15.09.17

  • Results

  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 20th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thurdsay 21st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 22nd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 25th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 27th
  • Phenol/Chloroform total RNA purification from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT
  • Protocol: Phenol/Chloroform purification of RNA
  • Participants: Jorge
  • Observations:
    • Four samples stored at -80 ºC labeled TRNA Lsh #1 27.09.17 and TRNA Lsh #2 27.09.17, TRNA Lsh #3 27.09.17, TRNA Lsh #4 27.09.17.

  • Results

  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 28th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 29th
  • FINA extraction for RNA with purified total RNA (TRNA Lsh #2-4 from 27.09.17)
  • Protocol: FINA Extraction for RNA
  • Participants: Jorge
  • Observations:
    • FINA was performed with 25 ul sample instead of 50 ul.
    • The extraction for TRNA #2 was performed as in protocol.
    • Before the FINA extraction, TRNA #3 was digested with DNase I according to DNase I Reaction Protocol
    • After the FINA extraction, the two eluate from TRNA #4 were digested with DNase I according to DNase I Reaction Protocol
  • First strand cDNA synthesis from E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT FINA-RNA eluate
  • Protocol: First strand cDNA synthesis
  • Participants: Jorge
  • Observations
    • pseq-Lsh-06rev was used as primer.
    • The eluates from the FINA extractions for RNA from 29.09.17 were used as samples.
    • From each pair of samples, one was used as a negative control (nuclease free water instead of MuLV-RT).
    • For all reactions, 6 ul sample were pipetted.
  • Q5-PCR for Lsh-fragment in E. coli Rosetta2 DE3 p2CT-His-MBP-Lsh_C13a_WT with cDNA samples from 29.09.17
  • Protocol: PCR amplification with Q5 Master Mix
  • Participants: Jorge
  • Observations:
    • pseq-Lsh-05fwd was used as forward primer.
    • pseq-Lsh-06rev was used as reverse primer.
    • Annealing was performed at 58 ºC.
    • The amplification step was 110 s long.
    • A 1.5% agarose gel was run in TAE-buffer at 110 V for 80 min to check for PCR-products.
    • Quick-Load Purple 2-Log DNA Ladder (N0550S) was used as ladder

  • Results

    • N: FINA extraction performed as in protocol.
    • NK: FINA extraction as in protocol. No RT in cDNA synthesis (negative control).
    • P: Digestion of DNA before FINA extraction.
    • PK: Digestion of DNA before FINA extraction. No RT in cDNA synthesis (negative control).
    • A: Digestion of DNA after FINA extraction.
    • AK: Digestion of DNA after FINA extraction. No RT in cDNA synthesis (negative control).
Monday 2nd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 3rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 4th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 5th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 6th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 9th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 11th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 12th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 13th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 16th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 17th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 18th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 19th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 20th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 23rd
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 24th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Wednesday 25th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Thursday 26th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Friday 27th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Monday 30th
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results
Tuesday 31st
  • Target
  • Protocol: protocol
  • Participants:
  • Observations:
    • Obs #1
    • Obs #2
  • readout
  • Protocol: Protocol
  • Participants:
  • Observations
  • Results