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<h1>15/04/22</h1>
<p>
<h2>Primer Design</h2>
Primer Design iILS1-9<br>
<br>
<br>

<h1>15/04/26</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20">Miniprep</a></h2>
<br>
<br>
<br>
<h1>15/06/03</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR</a> </h2>
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  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>PCR Mix </td>
        <td>PCR1</td>
        <td>PCR2</td>
        <td>PCR3</td>
        <td>PCR4</td>
        <td>PCR5</td>
        <td>PCR6</td>
    </tr>
    <tr>
        <td class='col'>0,5ul</td>
        <td class='col'>BB1 3108</td>
        <td class='col'>GFP1 3504</td>
        <td class='col'>BB2 3109</td>
        <td class='col'>GFP2 3504</td>
        <td class='col'>BB3 111</td>
        <td class='col'>GFP3 3504</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>ILS2</td>
        <td class='col'>ILS4</td>
        <td class='col'>ILS2</td>
        <td class='col'>ILS7</td>
        <td class='col'>ILS2</td>
        <td class='col'>ILS9</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>ILS3</td>
        <td class='col'>ILS5</td>
        <td class='col'>ILS6</td>
        <td class='col'>ILS5</td>
        <td class='col'>ILS8</td>
        <td class='col'>ILS5</td>
    </tr>
   <tr>
        <td class='col'>31,5ul</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
    </tr>
   <tr>
        <td class='col'>10ul</td>
        <td class='col'>Buffer</td>
        <td class='col'>Buffer</td>
        <td class='col'>Buffer</td>
        <td class='col'>Buffer</td>
        <td class='col'>Buffer</td>
        <td class='col'>Buffer</td>
    </tr>
   <tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPs</td>
    </tr>
   <tr>
        <td class='col'>2ul</td>
        <td class='col'>GXL Polymerase</td>
        <td class='col'>GXL Polymerase</td>
        <td class='col'>GXL Polymerase</td>
        <td class='col'>GXL Polymerase</td>
        <td class='col'>GXL Polymerase</td>
        <td class='col'>GXL Polymerase</td>
    </tr>

</table>

<br>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>Cycler Protocol (30x) </td>
        <td>PCR1,3,5</td>
        <td>PCR2,4,6</td>

    </tr>
    <tr>
        <td class='col'>98°C</td>
        <td class='col'>10s</td>
        <td class='col'>10s</td>

    </tr>
    <tr>
        <td class='col'>55°C</td>
        <td class='col'>15s</td>
        <td class='col'>15s</td>

    </tr>
    <tr>
        <td class='col'>68°C</td>
        <td class='col'>22s</td>
        <td class='col'>10s</td>

    </tr>
   <tr>
        <td class='col'>end cycle</td>
        <td class='col'></td>
        <td class='col'></td>

    </tr>
   <tr>
        <td class='col'>10°C</td>
        <td class='col'>store</td>
        <td class='col'>store</td>
  
    </tr>
</table>

<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR</a> </h2>
Repeat PCR of BB1, BB2<br>
Comments: unsatisfied yield for PCR1, PCR3 -> pcr of pcr  (3', 4') and repeat PCR with original template (1',2')
<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction</a></h2>
Gel extraction of PCR2,4,6
<br>
<br>
<br>

<h1>15/06/04</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR</a> </h2>
PCR of BB111 (BB3)
<br>
<br>
<br>

<h1>15/06/08</h1>
<p>
<h2>Gel electrophoresis</h2>
PCR1'-4'<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/5/52/MR_pic_ILS_08.06.2.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 1:</b> 

PCR1, 2, 3, 4

</figcaption>
</figure>
<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
PCR of GFP (PCR2,4,6)<br>
<br>

<h2>Gel electrophoresis</h2>
PCR2,4,6<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/5/52/MR_pic_ILS_08.06.2.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 2:</b> 

PCR1, 2, 3

</figcaption>
</figure>
<br>
<br>


<h1>15/06/09</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
Repeating PCR of PCR1, PCR3, PCR4<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Gel of PCR1,3,4<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/5/59/MR_pic_ILS_09.06.png" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 3:</b> 

PCR1, 3, 4<br>
PCR1 correct (longer band is the one)<br>
PCR3??

</figcaption>
</figure>
<br>
<br>

<h2>DpnI digest</h2>
Add 2ul of DpnI to the PCR, incubate for 30min @37°C<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol9">Gibson Assembly</a> </h2>
Gibson1: BB1+GFP1= pILS1 <br>
Gibson3: BB3+GFP3= pILS3 <br>
<br>

</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>  </td>
        <td>Gibson 1</td>
        <td>Gibson 2</td>

    </tr>
    <tr>
        <td class='col'>BB</td>
        <td class='col'>1,4ul</td>
        <td class='col'>1,5ul</td>

    </tr>
    <tr>
        <td class='col'>GFP</td>
        <td class='col'>3,23ul</td>
        <td class='col'>2,6ul</td>

    </tr>
    <tr>
        <td class='col'>H2OC</td>
        <td class='col'>5,4ul</td>
        <td class='col'>5,9ul</td>

    </tr>
   <tr>
        <td class='col'>Gibson MM</td>
        <td class='col'>10ul</td>
        <td class='col'>10ul</td>

    </tr>
    </tr>
</table>
<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol17">Transformation electrocompetent cells</a> </h2>
Transform 2ul Gibson mix of Gibson 1 and Gibson3 into NEB electrocompetent cells<br>
Recover 1h, plate 100ul<br>
<br>
<br>

<h1>15/06/11</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC </a> </h2>

</table>

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  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>  </td>
        <td>CPEC 1</td>
        <td>CPEC 3</td>

    </tr>
    <tr>
        <td class='col'>BB</td>
        <td class='col'>3,34ul</td>
        <td class='col'>3,78ul</td>

    </tr>
    <tr>
        <td class='col'>GFP</td>
        <td class='col'>0,69ul</td>
        <td class='col'>0,56ul</td>

    </tr>
    <tr>
        <td class='col'>H2OC</td>
        <td class='col'>5,97ul</td>
        <td class='col'>5,65ul</td>

    </tr>
    </tr>
</table>
<br>
<br>

<h2>Analytical digest</h2>

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  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td> BB2 </td>
        <td>4ul</td>

    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>3ul</td>

    </tr>
    <tr>
        <td class='col'>XbaI</td>
        <td class='col'>1ul</td>

    </tr>
    <tr>
        <td class='col'>SacI</td>
        <td class='col'>1ul</td>

    </tr>
    </tr>
        <td class='col'>Cutsmart</td>
        <td class='col'>1ul</td>

    </tr>
    </tr>
</table>
<br>
30min @37°C
<br>
<br>

<h2>Gel electrophoresis</h2>
Check the digest of BB2<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/2/2b/MR_pic_ILS_11.06.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 4:</b> 

Problem: Just one band visible; repeat with other enzymes and load undigested BB<br>

</figcaption>
</figure>
<br>
<br>
<br>

<h1>15/06/12</h1>
<p>
<h2>Analytical digest of BB2</h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td> Digest 1  </td>
        <td>   </td>
        <td> Digest 2  </td>
        <td>  </td>
        <td> Digest 3  </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB2</td>
        <td class='col'>4ul</td>
        <td class='col'>BB2</td>
        <td class='col'>7ul</td>
        <td class='col'>BB2</td>
        <td class='col'>4ul</td>

    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>3ul</td>
        <td class='col'></td>
        <td class='col'></td>
        <td class='col'>H2O</td>
        <td class='col'>2ul</td>

    </tr>
    <tr>
        <td class='col'>PvuII</td>
        <td class='col'>1ul</td>
        <td class='col'>EcoRI</td>
        <td class='col'>1ul</td>
        <td class='col'>EcoRI</td>
        <td class='col'>1ul</td>

    </tr>
    </tr>
        <td class='col'>PstI</td>
        <td class='col'>1ul</td>
        <td class='col'>PstI</td>
        <td class='col'>1ul</td>
        <td class='col'></td>
        <td class='col'></td>

    </tr>
    </tr>
        <td class='col'>Cutsmart</td>
        <td class='col'>1ul</td>
        <td class='col'>Cutsmart</td>
        <td class='col'>1ul</td>
        <td class='col'>Cutsmart</td>
        <td class='col'>1ul</td>

    </tr>
    </tr>
</table><br>
Incubate 30min @37°C
<br>
<br>


<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Check the different digests of BB2<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/5/59/MR_pic_ILS_12.06.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 5:</b> undigested - digest 1 - digest 2 - digest 3<br>
Something is wrong - they don'r show the correct size at all!!! Check registry and wells<br>

</figcaption>
</figure>
<br>
<br>
<br>

<h1>15/06/13</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent cells</a> </h2>
Transform following plasmids into NEB Turbo (Chemical competent):<br>
K823005 Promoter<br>
K823008 Promoter <br>
K823013 Promoter <br>
Gibson 1 <br>
Gibson3 <br>
CPEC1 <br>
CPEC3 <br>
<br>
<br>

<h1>15/06/15</h1>
<p>
<h2>Inoculation</h2>
K823005, K823008 & K823013 picked & inoculated into LB-medium <br>
Incubated @ 37 °C, 250 rpm
<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20">Miniprep</a> </h2>
Prep the Colonies of the ON<br><br>
From now on:<br>
K823005: BB04<br>
K823008: BB05<br>
K823013<br>
<br>
<br>

<h1>15/06/17</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td> </td>
        <td> PCR9  </td>
        <td> PCR10  </td>
    </tr>
    <tr>
        <td class='col'>0,5ul template</td>
        <td class='col'>BB K8230008 </td>
        <td class='col'>GFP4 (from PCR6)</td>
    </tr>
    <tr>
        <td class='col'>1ul Primer</td>
        <td class='col'>iILS8</td>
        <td class='col'>iILS9</td>
    </tr>
    <tr>
        <td class='col'>1ul Primer</td>
        <td class='col'>iILS2</td>
        <td class='col'>iILS5</td>
    </tr>
    </tr>
        <td class='col'>10ul</td>
        <td class='col'>buffer</td>
        <td class='col'>buffer</td>

    </tr>
    </tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPs</td>
        <td class='col'>dNTPS</td>
    </tr>
    </tr>
        <td class='col'>2ul</td>
        <td class='col'>Polymerase</td>
        <td class='col'>Polymerase</td>
    </tr>
    </tr>
        <td class='col'>31,5ul</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
    </tr>
    </tr>
</table><br><br>


<h2>Gel electrophoresis</h2>
Gel of Digest of the 06/16<br>
Lane1: GFP04, Lane 2: BB08
<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Extraction of BB08 and GFP04<br>
<br>
<br>

<h1>15/06/18</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC </a> </h2>
CPEC2 (pILS5)<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td> </td>
        <td> CPEC2  </td>
    </tr>
    <tr>
        <td class='col'>2,5ul</td>
        <td class='col'>BB08 </td>
    </tr>
    <tr>
        <td class='col'>4,29ul</td>
        <td class='col'>GFP04</td>
    </tr>
    <tr>
        <td class='col'>27,21</td>
        <td class='col'>H2O</td>
    </tr>
    </tr>
        <td class='col'>10ul</td>
        <td class='col'>buffer</td>
    </tr>
    </tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPs</td>
    </tr>
    </tr>
        <td class='col'>2ul</td>
        <td class='col'>Polymerase</td>
    </tr>
    </tr>

</table><br><br>
<br>

<h1>15/06/24</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
PCR for pILS4 and pILS6<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td> PCR Mix</td>
        <td> PCR7  </td>
        <td> PCR8  </td>
        <td> PCR11 </td>
        <td> PCR12 </td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>BB05 </td>
        <td class='col'>GFP</td>
        <td class='col'>BB13 </td>
        <td class='col'>GFP</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>Primer iILS2 </td>
        <td class='col'>Primer iILS5</td>
        <td class='col'>Primer iILS18 </td>
        <td class='col'>Primer iILS5</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>Primer iILS16 </td>
        <td class='col'>Primer iILS15</td>
        <td class='col'>Primer iILS115 </td>
        <td class='col'>Primer iILS17</td>
    </tr>
    </tr>
        <td class='col'>31ul</td>
        <td class='col'>H2O </td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
    </tr>
    </tr>
        <td class='col'>10ul</td>
        <td class='col'>buffer </td>
        <td class='col'>buffer</td>
        <td class='col'>buffer</td>
        <td class='col'>buffer</td>
    </tr>
    </tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPS </td>
        <td class='col'>dNTPS</td>
        <td class='col'>dNTPS</td>
        <td class='col'>dNTPS</td>
    </tr>
    </tr>
        <td class='col'>2ul</td>
        <td class='col'>Polymerase </td>
        <td class='col'>Polymerase</td>
        <td class='col'>Polymerase</td>
        <td class='col'>Polymerase</td>
    </tr>
    </tr>

</table><br><br>


<h2>Gel electrophoresis</h2>
Load the PCR on a gel to cut out<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Extract all PCRs <br>
<br>
<br>

<h1>15/06/29</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC </a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>pILS4</td>
        <td>   </td>
        <td>  pILS6 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB04</td>
        <td class='col'>0,2ul </td>
        <td class='col'>BB06</td>
        <td class='col'>0,8ul</td>
    </tr>
    <tr>
        <td class='col'>GFP4</td>
        <td class='col'>1,2ul </td>
        <td class='col'>GFP6</td>
        <td class='col'>0,9ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>32,62ul</td>
        <td class='col'>H2O</td>
        <td class='col'>32,35ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br>
Next day: Transformation - no colonies grew
<br>
<br>
<br>


<h1>15/07/01</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
PCR for pILS5<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>PCR Mix</td>
        <td> PCR9  </td>
        <td>  PCR10 </td>
    </tr>
    <tr>
        <td class='col'>0,5ul</td>
        <td class='col'>BB08 </td>
        <td class='col'>GFP</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>iILS8 </td>
        <td class='col'>iILS9</td>
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>iILS2</td>
        <td class='col'>iILS5</td>
    </tr>
    </tr>
        <td class='col'>31,5ul</td>
        <td class='col'>H2O</td>
        <td class='col'>H2O</td>
    </tr>
    </tr>
        <td class='col'>10ul</td>
        <td class='col'>buffer </td>
        <td class='col'>buffer</td>
    </tr>
    </tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPs </td>
        <td class='col'>dNTPs</td>
    </tr>
    </tr>
        <td class='col'>2ul</td>
        <td class='col'>Polymerase </td>
        <td class='col'>Polymerase</td>
    </tr>
    </tr>
</table><br>
<br>


<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
PCR of GFP4, GFP6, BB4, BB6<br>
Gel showes that BB6 was not succesful<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
extract BB4, GFP4, GFP6 from gel<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
Repeat PCR of BB6<br>
<br>
<br>

<h1>15/07/02</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC</a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>CPEC5</td>
        <td>   </td>
        <td>  CPEC4 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB4</td>
        <td class='col'>3,5ul </td>
        <td class='col'>BB04</td>
        <td class='col'>1,9ul</td>
    </tr>
    <tr>
        <td class='col'>GFP5</td>
        <td class='col'>5,1ul </td>
        <td class='col'>GFP4</td>
        <td class='col'>2,2ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>25,4ul</td>
        <td class='col'>H2O</td>
        <td class='col'>29,9ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br><br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent</a> </h2>
Transform CPECS into NEB (chemically)<br>
no colonies grew
<br>
<br>
<br>

<h1>15/07/06</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC </a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>CPEC5</td>
        <td>   </td>
        <td>  CPEC4 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB4</td>
        <td class='col'>3,5ul </td>
        <td class='col'>BB04</td>
        <td class='col'>1,9ul</td>
    </tr>
    <tr>
        <td class='col'>GFP5</td>
        <td class='col'>5,1ul </td>
        <td class='col'>GFP4</td>
        <td class='col'>2,2ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>25,4ul</td>
        <td class='col'>H2O</td>
        <td class='col'>29,9ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br>
use different cycler protocol<br>
<br>
<br>

<h1>15/07/07</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent</a> </h2>
Transform CPEC4, 5 into NEB turbo (chemical transformation)<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol10">Glycerol stocks</a></h2>
Prepare glycerol stocks (siGEM08, -siGEM11) of pILS4,5,6 of overnight cultures<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20">Miniprep</a> </h2>
Miniprep of Overnight Cultures<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </</h2>
Repeat PCR7-12<br>
<br>
<br>

<h1>15/07/08</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </</h2>
PCR of 7-12<br>
PCR7,8,9,10,12 were successful<br>
Repeat PCR11
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12">Gel Extraction</a> </h2>
Extract PCRs that worked<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC Standard</a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>CPEC4</td>
        <td>   </td>
        <td>  CPEC6 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB4</td>
        <td class='col'>0,41ul </td>
        <td class='col'>BB06</td>
        <td class='col'>0,37ul</td>
    </tr>
    <tr>
        <td class='col'>GFP4</td>
        <td class='col'>4,23ul </td>
        <td class='col'>GFP6</td>
        <td class='col'>9,26ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>29,35ul</td>
        <td class='col'>H2O</td>
        <td class='col'>24,37ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent</a> </h2>
Transform PEC5 and CPEC6 of the 6th into NEB chem comp. Use 5ul of DNA.<br>
No colonies for CPEC4 the next day<br>
Colonies for CPEC5.<br>
<br>

<h2>Inoculation</h2>
Inoculate 8 colonies of CPEC5 in 5ml LB-CAM and incubate @37°C for 6h.<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20"> Miniprep</a> </h2>
Prep the cultures.<br>
<br>

<h2>Analytical digest</h2>
Digest the 8 plasmids with PvuII<br>
<br>
<br>

<h1>15/07/10</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Extract PCR BB6, BB4, GFP4 and GFP6 out of Gel<br>
<br>

<h2>CPEC</h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>CPEC4</td>
        <td>   </td>
        <td>  CPEC6 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB4</td>
        <td class='col'>1,25ul </td>
        <td class='col'>BB06</td>
        <td class='col'>8ul</td>
    </tr>
    <tr>
        <td class='col'>GFP4</td>
        <td class='col'>0,42ul </td>
        <td class='col'>GFP6</td>
        <td class='col'>0,42ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>32,33ul</td>
        <td class='col'>H2O</td>
        <td class='col'>31,58ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br><br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol13">PCR </a> </h2>
New PCR of BB6<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol12"> Gel Extraction </a> </h2>
Extract BB6 BB6<br>
<br>


<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol5">CPEC </a> </h2>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>CPEC4</td>
        <td>   </td>
        <td>  CPEC6 </td>
        <td>  </td>
    </tr>
    <tr>
        <td class='col'>BB4</td>
        <td class='col'>2,4ul </td>
        <td class='col'>BB06</td>
        <td class='col'>1,3ul</td>
    </tr>
    <tr>
        <td class='col'>GFP4</td>
        <td class='col'>0,7ul </td>
        <td class='col'>GFP6</td>
        <td class='col'>0,8ul </td>
    </tr>
    <tr>
        <td class='col'>H2O</td>
        <td class='col'>30,9ul</td>
        <td class='col'>H2O</td>
        <td class='col'>31,9ul </td>
    </tr>
    </tr>
        <td class='col'>buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>buffer</td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul </td>
        <td class='col'>dNTPs</td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul </td>
        <td class='col'>Polymerase</td>
        <td class='col'>4ul</td>

    </tr>
    </tr>

</table><br><br>
<br>


<h1>15/07/11</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent</a> </h2>
Transform all previous CPECs into NEB turbo, chemical competent cells<br>
<br>
<br>

<h1>15/07/12</h1>
<p>
<h2>Inoculation</h2>
Pick 8 colonies of each construct and grow them in LB CAM<br>
<br>
<br>

<h1>15/07/13</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20">Miniprep</a> </h2>
Prep all the colonies from construct 4 and 6<br>
<br>

<h2>Analytical digest</h2>
Digest with PvuII<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/e/e7/MR_pic_ILS_13.07.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 6:</b> Lower bands look ok, but somehow there are additional long bands. Chromosomal DNA? Repeat Miniprep<br>

</figcaption>
</figure>
<br>
<br>
<br>

<h1>15/07/15</h1>
<p><h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol20">Miniprep</a> </h2>
Repeat Miniprep of colony 1-8 of construct 4 and 6<br>
<br>

<h2>Digest</h2>
Repeat digest with PvuII<br>
<br>

<h2>Overnight Cultures</h2>
Start ONC of 6.1 and 6.6<br>
<br>
<br>

<h1>15/07/16</h1>
<p>
<h2>Digest</h2>
New digest with new Minipreps with PvuII<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/e/e8/MR_pic_ILS_16.07_1.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 7:</b> Loaded also undigested plasmid; still weird long bands<br>
</figcaption>
<br>

</figure>
<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/a/ae/MR_pic_ILS_16.07_2.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 8:</b> Loaded also undigested plasmid; still weird long bands<br>
</figcaption>
</figure>
<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/9/90/MR_pic_ILS_16.07_3.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 9:</b> Loaded also undigested plasmid; still weird long bands<br>
</figcaption>
</figure>
<br>
<br>

<h2>Over night cultures</h2>
Start ONC of pILS5 for glycerol stock, 4.2, 4.4, 4.7, 6.1, 6.5<br>
<br>
<br>

<h1>15/07/17</h1>
<p>
<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol10">Glycerol stock </a</h2>
Prepare a glycerol stock of pILS5 (sIGEM20)and pILS4 (sIGEM21)<br>
<br>

<h2><a href="https://2015.igem.org/Team:Marburg/Protocols/Protocol16">Transformation chemocompetent</h2>
Transform pILS4 into Dh5alpha<br>
<br>


<h2>Miniprep</h2>
Prep 6.1 and 6.6<br>
<br>

<h2>Digest</h2>
Analytical digest of 6.1 and 6.6 with PvuII<br>
<br>


<h2>Sequencing</h2>
Send 6.1 and 6.6 for sequencing<br>
<br>
<br>

<h1>15/07/20</h1>
<p>
<h2>Transformation</h2>
Transform pILS4,5,6 into wt3110 and MG1655<br>
<br>
<br>

<h1>15/07/21</h1>
<p>
<h2>Over night culture</h2>
Pick colonies from pILS4,5,6 in wt3110 and MG1655 and start cultures<br>
<br>
<br>

<h1>15/07/22</h1>
<p>
<h2>Glycerol Stocks</h2>
WT3110: pILS4-6 - sIGEM 25-27<br>
MG1655: pILS4-6 - sIGEM 28-31<br>
<br>
<br>

<h1>15/07/27</h1>
<p>
<h2>Restreak all constructs </h2>
Restreak all strains on plates from glycerol stock<br>
<br>
<br>

<h1>15/08/11</h1>
<p>
<h2>Transformation</h2>
Transformation of E1010 and J23100 into NEB turbo<br>
J23100 wasn't succesful<br>
<br>
<br>

<h1>15/07/12</h1>
<p>
<h2>FACS</h2>
Measurement of 3 biological replicats and 3 technical replicats<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td>t=</td>
        <td>  dilution </td>
    </tr>
    <tr>
        <td class='col'>0h</td>
        <td class='col'>- </td>
    </tr>
    <tr>
        <td class='col'>1h</td>
        <td class='col'>- </td>
    </tr>
    <tr>
        <td class='col'>2h</td>
        <td class='col'>1:10</td>
    </tr>
    </tr>
        <td class='col'>3h</td>
        <td class='col'>1:100 </td>
    </tr>
    </tr>
        <td class='col'>4h</td>
        <td class='col'>1:100 </td>
    </tr>
    </tr>
        <td class='col'>5h</td>
        <td class='col'>1:100 </td>
    </tr>
    </tr>
        <td class='col'>6h</td>
        <td class='col'>1:100 </td>
    </tr>
    </tr>
        <td class='col'>6h</td>
        <td class='col'>1:100 </td>
    </tr>
    </tr>


</table><br>
<br>
<br>

<h1>15/07/13</h1>
<p>
<h2>Transformation</h2>
Repeat Transformation of J23100 in NEB turbo<br>
<br>
<br>

<h1>15/07/17</h1>
<p>
<h2>Transformation</h2>
Repeat Transformation of J23100 in NEB turbo<br>
<br>

<h2>PCR</h2>
PCR of BB7, BB8, BB9, BB10, T10, BB11, T11, BB12, T12<br>
<br>


<h2>Over night culture</h2>
ONC of E1010, pILS4, pILS5, pILS5 in DH5alpha<br>
<br>
<br>

<h1>15/08/18</h1>
<p>
<h2>Miniprep</h2>
Prep the cultures of E1010, pILS4, pILS5 and pILS6<br>
<br>


<h2>PCR</h2>
PCR of RFP7, RFP8, RFP9, RFP10, RFP11, RFP12<br>
<br>


<h2>Gel of PCRS</h2>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/3/38/MR_pic_ILS_18.08.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 10:</b> Weird bands<br>
</figcaption>
</figure>
<br>

<h2>DpnI digest</h2>
Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C<br>
<br>

<h2>PCR Purification</h2>
Purify BB7-9 and RFP7-9<br>
Low in concentration afterwards<br>
<br>

<h2>Gel extraction </h2>
Extraction of BB10, RFP10, Term10, BB11, RFP11, Term11, BB12, RFP12, Term12<br>
Low concentrations: repeat PCRs<br>
<br>


<h2>PCR</h2>
Repeat PCRs of BB7-12, RFP7-12, T10-12<br>
<br>

<h2>Analytical gel </h2>
Check BB7, RFP7, BB8, RFP8, BB9, RFP9 on gel<br>
<br>

<h2>DpnI digest</h2>
Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C<br>
<br>

<h2>Transformation</h2>
Transform J23100 into electrocompetent cells<br>
<br>
<br>


<h1>15/08/19</h1>
<p>
<h2>PCR Purification</h2>
Purify BB7, RFP7, BB8, RFP8, BB9, RFP9<br>
<br>

<h2>Gibson Assembly</h2>
15min @50°C<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td></td>
        <td>  BB </td>
        <td>  RFP </td>

    </tr>
    <tr>
        <td class='col'>7</td>
        <td class='col'>0,58ul </td>
        <td class='col'>0,91ul </td>
    </tr>
    <tr>
        <td class='col'>8</td>
        <td class='col'>0,8ul </td>
        <td class='col'>0,87ul</td>
    </tr>
    <tr>
        <td class='col'>9</td>
        <td class='col'>0,68ul</td>
        <td class='col'>0,74 </td>
    </tr>
    </tr>

</table><br><br>

<h2>Transformation</h2>
Transform the Gibson Assemblies into NEB turbo<br>
<br>


<h2>PCR</h2>
PCRs of BB10-12, RFP10-12 and Term 10-12<br>
<br>

<h2>Gel extraction</h2>
Extract BB10, RFP10, Term 10, BB11, RFP11, Term 11, BB12, RFP12, Term 12<br>
<br>

<h2>Over night culture</h2>
Start cultures of pILS4-6 in DH5alpha and MG1655<br>
<br>
<br>

<h1>15/08/20</h1>
<p>
<h2>Platereader experiment</h2>
Grow pILS4-6 in MG1655 and DH5alpha in M9 inplater reader<br>
Starting OD 0,01 in 96well plate, measure OD and GFP every 15min<br>
<br>

<h2>Over night cultures</h2>
Start cultures of pILS7.1-7.8, pILS7.1-8.8, pILS7.1-9.8 and J23100<br>
<br>

<h2>Transformation</h2>
Transform pILS9 into NEB turbo<br>
<br>
<br>

<h1>15/09/21</h1>
<p>
<h2>Miniprep</h2>
Prep the cultures of pILS7.1-7.8, pILS8.1-8.8 and pILS9.1<br>
<br>

<h2>PCR</h2>
Make the PCRS of Promoter 10-12<br>
<br>

<h2>Gel</h2>
no bands visible at expected length<br>
<br>
<br>

<h1>15/08/22</h1>
<p>
<h2>Digest</h2>
Digest pILS7.1-7.8, pILS8.1-8.8 and pILS9.1 with PvuII<br>
<figure style="text-align:center;">
  <img src="https://static.igem.org/mediawiki/2015/f/fd/MR_pic_ILS_21.08.jpg" width="200" alt="ILS" />
  <figcaption style="margin-top:5px;font-size:11pt;color:#606060;text-align:center;line-height:110%"><b>Figure 11:</b> pILS7.1, 8.3 and 9.1 look promising, send for sequencing<br>
</figcaption>
</figure>
<br>
<br>
<br>

<h1>15/08/24</h1>
<p>
<h2>Over night cultures</h2>
Start cultures of pILS9.2, pILS9.3, pILS9.4 and pILS9.5<br>
<br>

<h2>Transformation</h2>
Transform 0040, I20270 into DH5alpha and pILS9 (Gibson) in NEB turbo<br>
<br>
<br>

<h1>15/08/25</h1>
<p>
<h2>Restreak from glycerol stock</h2>
Restreak 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha on plates from glycerol stock - incubate for 16h<br>
<br>

<h2>Digestion of Promoter/h2>
Digest Promoter 10-12 with PvuII and EcoRI<<br>
<br>

<h2>Gel extraction</h2>
extract the band<br>
<br>

<h2>PCR</h2>
make a PCR on gel extracted digested promoter<br>
- no bands visible <br>
<br>
<br>

<h1>15/08/26</h1>
<p>
<h2>Over night cultures</h2>
Start cultures (8 biological replica) of 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha <br>
<br>

<h2>Sequencing</h2>
Sequencing of pILS7-9 showed point mutations<br>
<br>
<br>

<h1>15/08/27</h1>
<p>
<h2>Measurement with platereader for ILS submission</h2>
Dilution of each culture to OD of 0.5 (platereader) and measurement of fluorescence intensities<br>
<br>
<br>

<h1>15/08/28</h1>
<p>
<h2>Measurement with platereader for ILS submission</h2>
Dilution of each culture to OD of 0.5 (cuvette) and measurement of fluorescence intensities<br>
<br>
<br>


<h1>15/08/29</h1>
<p>
<h2>PCR </h2>
Amplify parts for pILS7-9<br>
<br>

<h2>PCR Purification</h2>
Purification of the PCRs<br>
<br>

<h2>Gibson Assembly</h2>
Assembly of pILS7-9<br>
<br>

<h2>Transformation</h2>
Transform Gibson Assembly into NEB Turbo<br>
<br>
<br>


<h1>15/08/30</h1>
<p>
<h2>Over night cultures</h2>
Start cultures of Gibson Assemblies and J23100<br>
<br>
<br>

<h1>15/08/31</h1>
<p>
<h2>Miniprep</h2>
Prep the cultures of pILS7-9, J23100<br>
<br>

<h2>Sequencing</h2>
Send pILS7-9 for sequencing:<br>
Constructs: 2.7.2, 2.8.2, 2.9.3, 3.7.1, 3.7.2, 3.8.1, 3.8.3, 3.9.2, 3.9.3<br>
<br>


<h2>PCR</h2>
Fuse two primers to promoter by PCR<br>
</table>

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</head>


  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td></td>
        <td>  Parts </td>

    </tr>
    <tr>
        <td class='col'>5ul</td>
        <td class='col'>iILS30 </td>
    </tr>
    <tr>
        <td class='col'>5ul</td>
        <td class='col'>iILS31 </td>
    </tr>
    <tr>
        <td class='col'>2ul</td>
        <td class='col'>Polymerase</td>
    </tr>
    </tr>
        <td class='col'>4ul</td>
        <td class='col'>dNTPs</td>
    </tr>
    </tr>
        <td class='col'>10ul</td>
        <td class='col'>buffer</td>
    </tr>
    </tr>
</table><br>
<br>

<h2>PCR Purification</h2>
Purify the fragment of the Primer PCR<br>
<br>


<h2>CPEC</h2>
</table>

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  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td></td>
        <td>  10.1 </td>
        <td>10.3</td>
        <td>  11.1 </td>
        <td>11.3</td>
        <td>  12.1 </td>
        <td>  12.3 </td>
    </tr>
    <tr>
        <td class='col'>BB</td>
        <td class='col'>0,7ul </td>
        <td class='col'>0,7ul</td>
        <td class='col'>0,65ul </td>
        <td class='col'>0,65ul</td>
        <td class='col'>1,14ul </td>
        <td class='col'>1,14ul</td>
    </tr>
    <tr>
        <td class='col'>RFP</td>
        <td class='col'>0,32ul </td>
        <td class='col'>0,32ul</td>
        <td class='col'>0,208ul </td>
        <td class='col'>0,208ul</td>
        <td class='col'>0,26ul </td>
        <td class='col'>0,26ul</td>
    </tr>
    <tr>
        <td class='col'>Promoter</td>
        <td class='col'>0,32ul </td>
        <td class='col'>0,46ul</td>
        <td class='col'>0,32ul </td>
        <td class='col'>0,46ul</td>
        <td class='col'>0,32ul </td>
        <td class='col'>0,46ul</td>
    </tr>
    </tr>
        <td class='col'>Terminator</td>
        <td class='col'>3,81ul </td>
        <td class='col'>3,81ul</td>
        <td class='col'>3,81ul </td>
        <td class='col'>3,81ul</td>
        <td class='col'>3,81ul </td>
        <td class='col'>3,81ul</td>
    </tr>
    </tr>
        <td class='col'>H2O</td>
        <td class='col'>28,85ul </td>
        <td class='col'>28,71ul</td>
        <td class='col'>28,94ul </td>
        <td class='col'>28,81ul</td>
        <td class='col'>28,48ul </td>
        <td class='col'>28,34ul</td>
    </tr>
    </tr>
        <td class='col'>dNTPS</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul</td>
    </tr>
    </tr>
        <td class='col'>Buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul</td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul</td>
    </tr>
    </tr>
</table><br>
<br>
<br>

<h1>15/09/01</h1>
<p>
<h2>Sequencing</h2>
Sequencing of pILS2.8.2 and pILS3.7.1 were successful<br>
For pILS9 still no positive clone - send more samples for sequencing<br>
<br>


<h2>Transformation</h2>
Transformation of CPEC10.1-10.4, 11.1-11.4 and 12.1-12.4 into NEB (with 1ul and 2ul)<br>
<br>

<h2>PCR</h2>
Repeat PCR of primer bindin iILS30, iILS31<br>
<br>


<h2>PCR Purification</h2>
Purify the Primer PCR<br>
<br>

<h2>CPEC</h2>
</table>

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  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td></td>
        <td>  10.4 </td>
        <td>11.4</td>
        <td>  12.4 </td>
 
    </tr>
    <tr>
        <td class='col'>BB</td>
        <td class='col'>4,17ul </td>
        <td class='col'>17,13ul</td>
        <td class='col'>14,43ul </td>
    <tr>
        <td class='col'>RFP</td>
        <td class='col'>7,33ul </td>
        <td class='col'>7,33ul</td>
        <td class='col'>7,33ul </td>
    </tr>
    <tr>
        <td class='col'>Promoter</td>
        <td class='col'>0,59ul </td>
        <td class='col'>0,59ul</td>
        <td class='col'>0,59ul </td>
    </tr>
    </tr>
        <td class='col'>Terminator</td>
        <td class='col'>0,56ul </td>
        <td class='col'>0,56ul</td>
        <td class='col'>0,56ul </td>
    </tr>
    </tr>
        <td class='col'>H2O</td>
        <td class='col'>21,56ul </td>
        <td class='col'>7,8ul</td>
        <td class='col'>11,08ul </td>

    </tr>
    </tr>
        <td class='col'>dNTPS</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul</td>
        <td class='col'>4ul </td>
    </tr>
    </tr>
        <td class='col'>Buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul</td>
        <td class='col'>10ul </td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul</td>
        <td class='col'>2ul </td>
    </tr>
    </tr>
</table><br>
<br>
<br>

<h1>15/09/02</h1>
<p>
<h2>Transformation</h2>
Trafo of constr. 10,4+11,4+12,4 in NEB Turbo<br>
<br>
<h2>PCR</h2>
PCR of iILS30+iILS31<br>
<br>
<h2>PCR Cleanup</h2>
PCR-Clean up of iILS30+iILS31<br>
<br>
<h2>CPEC</h2>
CPEC of construct 5 <br>
<br>

<h2>Sequencing</h2>
Send pILS2.9.1 and 2.9.2 for sequencing<br>
<br>

<h2>Over night cultures</h2>
Start cultures for 9, 10.1, 11.1, 12.1<br>
<br><br>

<h1>15/09/03</h1>
<p>
<h2>Miniprep of ONC 9+10+10</h2>
Analytical Digest with PvuII<br>
<br>

<h2>Sequencing</h2>
Sequencing of 12.3, 11.3, 10.3, 9.3 <br>
<br><br>

<h1>15/09/07</h1>
<p>
<h2>Sequencing</h2>
10.3.1 was correct -> pILS10<br>
11.3.1 was correct -> pILS11<br>
Transformation of pILS10, pILS11 in DH5alpha cells <br>
<br>

<h2>New Method for 9/12</h2>

  <table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
  <tr style="background-color:#f66300;">
        <td></td>
        <td>  PCR 9.1 </td>
        <td>  PCR 9.2 </td>
        <td>  PCR 12.1 </td>
        <td>  PCR 12.2 </td>
 
    </tr>
    <tr>
        <td class='col'>0,5 ul Template</td>
        <td class='col'>pILS11</td>
        <td class='col'>pILS6 </td>
        <td class='col'>pILS6</td>
        <td class='col'>pILS8 </td>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>Primer ILS 17 </td>
        <td class='col'>Primer ILS 18 </td>
        <td class='col'>Primer ILS 21 </td>
        <td class='col'>Primer ILS 24 </td>
        
    </tr>
    <tr>
        <td class='col'>1ul</td>
        <td class='col'>Primer ILS 20 </td>
        <td class='col'>Primer ILS 22 </td>
        <td class='col'>Primer ILS 22 </td>
        <td class='col'>Primer ILS 20 </td>
    </tr>
    </tr>
        <td class='col'>31,5 ul</td>
        <td class='col'>H2O </td>
         <td class='col'>H2O </td>
         <td class='col'>H2O </td>
         <td class='col'>H2O </td>

    </tr>
    </tr>
        <td class='col'>dNTPS</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul</td>
        <td class='col'>4ul </td>
        <td class='col'>4ul </td>
    </tr>
    </tr>
        <td class='col'>Buffer</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul</td>
        <td class='col'>10ul </td>
        <td class='col'>10ul </td>
    </tr>
    </tr>
        <td class='col'>Polymerase</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul</td>
        <td class='col'>2ul </td>
        <td class='col'>2ul </td>
    </tr>
    </tr>
</table><br><br>
<br>


<h2>dNPI-Digest</h2>
1ul Enzyme, 30 min at 37°C<br>
<br>
after the Thermo Fischer PCR-Clean up Kit Protokoll<br>
<br>

<h2>Gibson Assembly</h2>

<h2>Transformation in NEB turbo cells</h2>
<br><br>

<h2>Over night culture</h2>
of pILS4,5,6 in DH5alpha for quantitiative proteomics<br><br>


<h1>15/09/08</h1>
<p>
<h2>Over night culture</h2>
of pILS7,8 in DH5alpha<br><br>
<h2>Over night culture</h2>
of pILS9,12 from Gibson Assembly <br><br>
<br>



<h1>15/09/09</h1>
<p>
<h2>Preperation of Glycerolstock</h2>
pILS 7 in DH5alpha -> SiGEM 51<br>
pILS 8 in DH5alpha -> SiGEM 52<br>
<br>

<h2>Miniprep</h2>
of ONC 9,12 from Gibson Assembly<br>
<h2>analytical-Digest of 9,12</h2>
1ul PvuII, 30 min at 37°C<br>
<br>

<h2>Sequencing</h2>
of pILS9,12 from Gibson Assembly<br>

<h2>Over Night Cultures</h2>
of pILS4,5,6<br>
<br><br>

<h1>15/09/10</h1>
<p>
<h2>Start of Evolution Study </h2>
ONC of pILS 4,5,6 in DH5alpha in 3 biologcal replica <br>
<br>
<h2>Preperation of samples 4,5,6 for Proteomics </h2>
Sonication of ONC pILS4,5,6 for 15s, 15 Cycles <br>
-> to Uwe Linner <br>
<br>
<h2>Preperation of samples 4,5,6 for Microscopy </h2>
measured with Oliver and Anne <br>
<br><br>

<h1>15/09/11</h1>
<p>
<h2>8am: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br>

<h2>8pm: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br><br>

<h1>15/09/12</h1>
<p>
<h2>8am: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br>

<h2>8pm: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br>

<h2>Transfomation</h2>
of piLS7,8,10,11, J0445, J20270 in DH5alpha and MG1655<br>
<br><br>

<h1>15/09/13</h1>
<p>
<h2>8am: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br>

<h2>8pm: FACS measurement </h2>
2ml Tethering Buffer +100 ul Sample <br>
afterwards dilued to an OD of 0,05<br>
Incubated at 30° for 12h<br>
<br><br>

<h1>DATE</h1>
<p>
<h2>Text</h2>
lalala<br>
<br>


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