September

From 2008.igem.org

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[[image:11.09.08 Test Bindung Origami to alexa-Freigem08.jpg |880px]]<br>
[[image:11.09.08 Test Bindung Origami to alexa-Freigem08.jpg |880px]]<br>
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<h3>09-12-2008</h3>
<h3>09-12-2008</h3>
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<html>
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'''1) Origami with NIP and fluorophor for the binding measurement'''
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<head>
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  <meta content="text/html; charset=ISO-8859-1"
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We had to produce some new origami for our next binding measurements.
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http-equiv="content-type">
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  <title></title>
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* Origami with NIP and fluorophor
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</head>
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<body>
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* Origami only with fluorophor (without NIP); negative control
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<p class="MsoNormal"><b style=""><span
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-
style="" lang="EN-GB">1) Origami with NIP and
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see at the protocol from 07-24-2008
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fluorophor for the
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-
binding measurement</span><u><span style=""
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'''2) Origami for the Calciummeasurement'''
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lang="EN-GB"><o:p></o:p></span></u></b></p>
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<p class="MsoNormal"><span style="" lang="EN-GB">We
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* Origami with NIP
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had to
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-
produce some new origami for our next binding measurements.<o:p></o:p></span></p>
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* Origami without NIP (negative control)
-
<ul>
+
 
-
  <li><span style="" lang="EN-GB"><span
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see at the protocol from 07-24-2008
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style=""></span></span><span
+
 
-
style="font-family: Wingdings;" lang="EN-GB"></span><span
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To increase the concentration of origami we also made to probes with the double amount
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style="" lang="EN-GB">Origami with NIP and fluorophor<o:p></o:p></span></li>
+
 
-
</ul>
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ingredients of the protocol from 07.24.2008
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<ul>
+
-
  <li><span style="" lang="EN-GB"><span
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<table border="1" rules="all">
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style=""></span></span><span
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style="font-family: Wingdings;" lang="EN-GB"></span><span
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<tr>
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style="" lang="EN-GB">Origami only with fluorophor
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<th></th>
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(without NIP);
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<td> Origami with NIP (6x (1:5)) [µl] </td>
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negative control<o:p></o:p></span></li>
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<td> Origami without NIP (6x (1:5)) [µl] </td>
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</ul>
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</tr>
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<p class="MsoNormal"><span style="" lang="EN-GB">see
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<tr>
-
at the
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<td> Oligos-Pool </td>
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protocol from </span><st1:date year="2008" day="24"
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<td align="center">43,68</td>
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month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
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<td align="center">43,68</td>
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style="" lang="EN-GB"><o:p></o:p></span></p>
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</tr>
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<p class="MsoNormal"><span style="" lang="EN-GB"><o:p></o:p></span><b
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<tr>
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style=""><span style="" lang="EN-GB">2)
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<td> remainders </td>
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Origami for the Calciummeasurement</span><u><span
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<td align="center">2,4</td>
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style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
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<td align="center">2,4</td>
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<ul>
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</tr>
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  <li><span style="" lang="EN-GB"><span
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<tr>
-
style=""></span></span><span
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<td> MgAc </td>
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style="font-family: Wingdings;" lang="EN-GB"></span><span
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<td align="center">1</td>
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style="" lang="EN-GB">Origami with NIP<o:p></o:p></span></li>
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<td align="center">1</td>
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</ul>
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</tr>
-
<ul>
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<tr>
-
  <li><span style="" lang="EN-GB"><span
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<td> Phage DNA (448,4 mg/µl) </td>
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style=""></span></span><span
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<td align="center">33,6</td>
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style="font-family: Wingdings;" lang="EN-GB"></span><span
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<td align="center">33,6</td>
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style="" lang="EN-GB">Origami without NIP (negative
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</tr>
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control)<o:p></o:p></span></li>
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<tr>
-
</ul>
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<td> NIP-Oligo </td>
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<p class="MsoNormal"
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<td align="center">1,68</td>
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style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
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<td align="center">----</td>
-
style="" lang="EN-GB">see at the
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</tr>
-
protocol from </span><st1:date year="2008" day="24"
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<tr>
-
month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
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<td> Pool oligo without fluorophor </td>
-
style="" lang="EN-GB"><o:p></o:p></span></p>
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<td align="center">0,72</td>
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<p class="MsoNormal"
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<td align="center">0,72</td>
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style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
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</tr>
-
style="" lang="EN-GB">To increase the concentration
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<tr>
-
of
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<td> Oligo without NIP </td>
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origami we also made to probes with the double amount<o:p></o:p></span></p>
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<td align="center">----</td>
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<p class="MsoNormal"
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<td align="center">1,68</td>
-
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
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</tr>
-
style="" lang="EN-GB">ingredients of the protocol
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-
from </span><st1:date year="2008" day="24"
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-
month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
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-
style="" lang="EN-GB"><o:p></o:p></span></p>
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-
<table class="MsoTableGrid"
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style="border: medium none ; border-collapse: collapse;"
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-
border="1" cellpadding="0" cellspacing="0">
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  <tbody>
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    <tr style="">
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-
      <td
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style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 107.4pt;"
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valign="top" width="143">
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      <p class="MsoNormal"><span style=""
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-
lang="EN-GB"><o:p>&nbsp;</o:p></span></p>
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      </td>
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      <td
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style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
+
-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">Origami with NIP (6x (1:5)) [&micro;l]<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
+
-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">Origami without NIP (6x (1:5)) [&micro;l]<o:p></o:p></span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr style="">
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
+
-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">Oligos-Pool<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">43,68<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
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-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">43,68<o:p></o:p></span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr style="">
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
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-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">remainders<o:p></o:p></span></p>
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-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">2,4<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">2,4<o:p></o:p></span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr style="">
+
-
      <td
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-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
+
-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">MgAc<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">1<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
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-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
+
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align="right"><span style="" lang="EN-GB">1<o:p></o:p></span></p>
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-
      </td>
+
-
    </tr>
+
-
    <tr style="">
+
-
      <td
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-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
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-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">Phage DNA (448,4 mg/&micro;l)<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
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-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
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align="right"><span style="" lang="EN-GB">33.6<o:p></o:p></span></p>
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      </td>
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-
      <td
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style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
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-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
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align="right"><span style="" lang="EN-GB">33.6<o:p></o:p></span></p>
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-
      </td>
+
-
    </tr>
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-
    <tr style="">
+
-
      <td
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style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
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valign="top" width="143">
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      <p class="MsoNormal"><span style=""
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-
lang="EN-GB">NIP-Oligo<o:p></o:p></span></p>
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-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">1,68<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
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valign="top" width="144">
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      <p class="MsoNormal"><span style=""
+
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lang="EN-GB">---------------------<o:p></o:p></span></p>
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-
      </td>
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-
    </tr>
+
-
    <tr style="">
+
-
      <td
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-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
+
-
      <p class="MsoNormal"><span style=""
+
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lang="EN-GB">Pool oligo without fluorophor<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">0,72<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">0,72<o:p></o:p></span></p>
+
-
      </td>
+
-
    </tr>
+
-
    <tr style="">
+
-
      <td
+
-
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 107.4pt;"
+
-
valign="top" width="143">
+
-
      <p class="MsoNormal"><span style=""
+
-
lang="EN-GB">Oligo without NIP<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 90pt;"
+
-
valign="top" width="120">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">-------------------<o:p></o:p></span></p>
+
-
      </td>
+
-
      <td
+
-
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0cm 5.4pt; width: 108pt;"
+
-
valign="top" width="144">
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-
      <p class="MsoNormal" style="text-align: right;"
+
-
align="right"><span style="" lang="EN-GB">1,68<o:p></o:p></span></p>
+
-
      </td>
+
-
    </tr>
+
-
  </tbody>
+
</table>
</table>
-
<p class="MsoNormal"
 
-
style="margin-left: 35.4pt; text-indent: -35.4pt;"><b
 
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style=""><span style="" lang="EN-GB"><o:p></o:p></span><span
 
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style="" lang="EN-GB">3) Master cycler</span><u><span
 
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style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
 
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<p class="MsoNormal"
 
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style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 
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style="" lang="EN-GB">The origamis were produced in
 
-
the
 
-
mastercycler as explained before.<o:p></o:p></span></p>
 
-
<p class="MsoNormal"
 
-
style="margin-left: 35.4pt; text-indent: -35.4pt;"><b
 
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style=""><o:p>&nbsp;</o:p><span style=""
 
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lang="EN-GB">4) Purification of the DNA Origami</span><u><span
 
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style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
 
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<p class="MsoNormal"
 
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style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 
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style="" lang="EN-GB">Was done as before<o:p></o:p></span></p>
 
-
</body>
 
-
</html>
 
-
'''5) Digestion of CMV+Rluc''' (Sabine)<br>
+
'''3) Master cycler'''
 +
 
 +
The origamis were produced in the mastercycler as explained before.
 +
 
 +
'''4) Purification of the DNA Origami'''
 +
 
 +
Was done as before
 +
 
 +
'''5) Digestion of CMV+Rluc (Sabine)'''
Digestion with EcoRV und FspI (3h at 37°C)
Digestion with EcoRV und FspI (3h at 37°C)
-
<ul>
+
 
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<li> 5µl plasmid
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* 5µl plasmid
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<li> 10µl H2O
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* 10µl H2O
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<li> 0,5µl enzyme
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* 0,5µl enzyme
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<li> 2,5µl buffer (2)
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* 2,5µl buffer (2)
-
<li> 0,5µl BSA
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* 0,5µl BSA
-
</ul>
+
 
[[image:Verdau_CMVRluc_EcoRV_FspI_klein.jpg]]<br>
[[image:Verdau_CMVRluc_EcoRV_FspI_klein.jpg]]<br>

Revision as of 20:01, 9 October 2008


Freiburg2008 small header.gif



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CoLABoration
__september



09-10-2008


CMV PCR (Sabine)
-PCR with primer for the CMV promotor; as DNA template the CMV+RLuc construct from the Ljubljana group (isolated from the parts collection 2007) was used.

For a 50 µl reaction:
40,4 µl H2O
5 µl buffer (10x)
1,5 µl FWD Primer (15pmol)(
1,5 µl REV Primer (15pmol)
1 µl dNTP (10mM)
0,1 µl DNA template
0,5 µl Pfu Polymerase

The settings for the PCR machine are the following:
1. T=94°C 00:02:00
2. T=94°C 00:00:30
3. T=62°C 00:00:30
4. T=72°C 00:01:00
5. GOTO 2 REP 29
6. T=72°C 00:10:00
7. HOLD 6°C

No product was received.

09-11-2008

11.09.08 Test Fura staining-Freigem08.jpg

11.09.08 Test Bindung Origami to alexa-Freigem08.jpg



09-12-2008

1) Origami with NIP and fluorophor for the binding measurement

We had to produce some new origami for our next binding measurements.

  • Origami with NIP and fluorophor
  • Origami only with fluorophor (without NIP); negative control

see at the protocol from 07-24-2008

2) Origami for the Calciummeasurement

  • Origami with NIP
  • Origami without NIP (negative control)

see at the protocol from 07-24-2008

To increase the concentration of origami we also made to probes with the double amount

ingredients of the protocol from 07.24.2008

Origami with NIP (6x (1:5)) [µl] Origami without NIP (6x (1:5)) [µl]
Oligos-Pool 43,68 43,68
remainders 2,4 2,4
MgAc 1 1
Phage DNA (448,4 mg/µl) 33,6 33,6
NIP-Oligo 1,68 ----
Pool oligo without fluorophor 0,72 0,72
Oligo without NIP ---- 1,68

3) Master cycler

The origamis were produced in the mastercycler as explained before.

4) Purification of the DNA Origami

Was done as before

5) Digestion of CMV+Rluc (Sabine) Digestion with EcoRV und FspI (3h at 37°C)

  • 5µl plasmid
  • 10µl H2O
  • 0,5µl enzyme
  • 2,5µl buffer (2)
  • 0,5µl BSA

File:Verdau CMVRluc EcoRV FspI klein.jpg

6) CMV PCR (Sabine)
-the same PCR with different annealing temperatures using a gradient between 58°C and 62°C (optimal annealing temperature: 62°C)
-again, no products were gained

09-15-2008

CMV-PCR (Sabine)
-another effort to gain the CMV-Promotor via PCR, this time with different polymerases: Taq Polymerase and a Mix.
-one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc)
-products in the approach with taq polymerase as well as in the approach with the Mix. 3 bands from each approach were cut out

09-16-2008

Gel purification (Sabine)
-gel purification of the PCR products

Digestion of the PCR products and the transfectionvector (Sabine, Kathrin)
-digestion with EcoRI and PstI
-ligation

09-18-2008

Transformation (Sabine)
-transformation with the ligation product.

09-19-2008

Transformation (Sabine)
-no colonies on the plates.

09-20-2008

Digestion of the PCR products and the transfection-vector (Sabine)
-the restriction-enzymes XbaI and SpeI were used

Gel purification and ligation (Sabine)
-digestion of PCR products and vector

09-21-2008

Transformation of the ligation (Sabine)
-RV 308 cells were transformed with 10 µl of the ligation

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