September

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{{Freiburg2008_Main|
{{Freiburg2008_Main|
Content=
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<div style="font-size:18pt;">
 +
<font face="Arial Rounded MT Bold" style="color:#010369">__september</font></div>
 +
<br>
 +
<br>
 +
__NOTOC__
 +
<h3>09-10-2008</h3>
 +
<br>
 +
'''CMV PCR'''<br>
 +
I did a PCR with primer for the CMV promotor. As DNA template I used the CMV+RLuc construct from the Ljubljana group which I isolated from the parts collection 2007.<br>
 +
 
 +
For a 50 µl reaction I used:<br>
 +
40,4 µl H2O<br>
 +
5 µl buffer (10x)<br>
 +
1,5 µl FWD Primer (15pmol)(<br>
 +
1,5 µl REV Primer (15pmol) <br>
 +
1 µl dNTP (10mM)<br>
 +
0,1 µl DNA template<br>
 +
0,5 µl Pfu Polymerase<br>
 +
 
 +
The settings for the PCR machine are the following:<br>
 +
1. T=94°C  00:02:00<br>
 +
2. T=94°C  00:00:30<br>
 +
3. T=62°C  00:00:30<br>
 +
4. T=72°C  00:01:00<br>
 +
5. GOTO 2  REP 29<br>
 +
6. T=72°C  00:10:00<br>
 +
7. HOLD  6°C<br>
 +
 
 +
I got no product.
 +
 
 +
<h3>09-11-2008</h3>
 +
[[image:11.09.08 Test Fura staining-Freigem08.jpg |880px]]<br>
 +
<br>
 +
[[image:11.09.08 Test Bindung Origami to alexa-Freigem08.jpg |880px]]<br>
 +
 
 +
 
 +
<h3>09-12-2008</h3>
 +
 
 +
<html>
 +
<head>
 +
  <meta content="text/html; charset=ISO-8859-1"
 +
http-equiv="content-type">
 +
  <title></title>
 +
</head>
 +
<body>
 +
<p class="MsoNormal"><b style=""><span
 +
style="" lang="EN-GB">1) Origami with NIP and
 +
fluorophor for the
 +
binding measurement</span><u><span style=""
 +
lang="EN-GB"><o:p></o:p></span></u></b></p>
 +
<p class="MsoNormal"><span style="" lang="EN-GB">We
 +
had to
 +
produce some new origami for our next binding measurements.<o:p></o:p></span></p>
 +
<ul>
 +
  <li><span style="" lang="EN-GB"><span
 +
style=""></span></span><span
 +
style="font-family: Wingdings;" lang="EN-GB"></span><span
 +
style="" lang="EN-GB">Origami with NIP and fluorophor<o:p></o:p></span></li>
 +
</ul>
 +
<ul>
 +
  <li><span style="" lang="EN-GB"><span
 +
style=""></span></span><span
 +
style="font-family: Wingdings;" lang="EN-GB"></span><span
 +
style="" lang="EN-GB">Origami only with fluorophor
 +
(without NIP);
 +
negative control<o:p></o:p></span></li>
 +
</ul>
 +
<p class="MsoNormal"><span style="" lang="EN-GB">see
 +
at the
 +
protocol from </span><st1:date year="2008" day="24"
 +
month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
 +
style="" lang="EN-GB"><o:p></o:p></span></p>
 +
<p class="MsoNormal"><span style="" lang="EN-GB"><o:p></o:p></span><b
 +
style=""><span style="" lang="EN-GB">2)
 +
Origami for the Calciummeasurement</span><u><span
 +
style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
 +
<ul>
 +
  <li><span style="" lang="EN-GB"><span
 +
style=""></span></span><span
 +
style="font-family: Wingdings;" lang="EN-GB"></span><span
 +
style="" lang="EN-GB">Origami with NIP<o:p></o:p></span></li>
 +
</ul>
 +
<ul>
 +
  <li><span style="" lang="EN-GB"><span
 +
style=""></span></span><span
 +
style="font-family: Wingdings;" lang="EN-GB"></span><span
 +
style="" lang="EN-GB">Origami without NIP (negative
 +
control)<o:p></o:p></span></li>
 +
</ul>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 +
style="" lang="EN-GB">see at the
 +
protocol from </span><st1:date year="2008" day="24"
 +
month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
 +
style="" lang="EN-GB"><o:p></o:p></span></p>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 +
style="" lang="EN-GB">To increase the concentration
 +
of
 +
origami we also made to probes with the double amount<o:p></o:p></span></p>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 +
style="" lang="EN-GB">ingredients of the protocol
 +
from </span><st1:date year="2008" day="24"
 +
month="7"><span style="" lang="EN-GB">07-24-2008</span></st1:date><span
 +
style="" lang="EN-GB"><o:p></o:p></span></p>
 +
<table class="MsoTableGrid"
 +
style="border: medium none ; border-collapse: collapse;"
 +
border="1" cellpadding="0" cellspacing="0">
 +
  <tbody>
 +
    <tr style="">
 +
      <td
 +
style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 107.4pt;"
 +
valign="top" width="143">
 +
      <p class="MsoNormal"><span style=""
 +
lang="EN-GB"><o:p>&nbsp;</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: 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">
 +
      <p class="MsoNormal" style="text-align: right;"
 +
align="right"><span style="" lang="EN-GB">43,68<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;"
 +
valign="top" width="144">
 +
      <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">
 +
      <p class="MsoNormal"><span style=""
 +
lang="EN-GB">remainders<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">
 +
      <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">
 +
      <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
 +
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">
 +
      <p class="MsoNormal" style="text-align: right;"
 +
align="right"><span style="" lang="EN-GB">1<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">
 +
      <p class="MsoNormal" style="text-align: right;"
 +
align="right"><span style="" lang="EN-GB">1<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">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;"
 +
valign="top" width="120">
 +
      <p class="MsoNormal" style="text-align: right;"
 +
align="right"><span style="" lang="EN-GB">33.6<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">
 +
      <p class="MsoNormal" style="text-align: right;"
 +
align="right"><span style="" lang="EN-GB">33.6<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">NIP-Oligo<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">
 +
      <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;"
 +
valign="top" width="144">
 +
      <p class="MsoNormal"><span style=""
 +
lang="EN-GB">---------------------<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">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">
 +
      <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">
 +
      <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">
 +
      <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">
 +
      <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>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><b
 +
style=""><span style="" lang="EN-GB"><o:p></o:p></span><span
 +
style="" lang="EN-GB">3) Master cycler</span><u><span
 +
style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 +
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
 +
style=""><o:p>&nbsp;</o:p><span style=""
 +
lang="EN-GB">4) Purification of the DNA Origami</span><u><span
 +
style="" lang="EN-GB"><o:p></o:p></span></u></b></p>
 +
<p class="MsoNormal"
 +
style="margin-left: 35.4pt; text-indent: -35.4pt;"><span
 +
style="" lang="EN-GB">Was done as before<o:p></o:p></span></p>
 +
</body>
 +
</html>
 +
 
 +
'''5) Digestion of CMV+Rluc'''<br>
 +
Digestion with EcoRV und FspI(3h at 37°C)
 +
<ul>
 +
<li> 5µl plasmid
 +
<li> 10µl H2O
 +
<li> 0,5µl enzyme
 +
<li> 2,5µl buffer (2)
 +
<li> 0,5µl BSA
 +
</ul>
 +
[[image:Verdau_CMVRluc_EcoRV_FspI_klein.jpg]]<br>
 +
 
 +
'''6) CMV PCR'''<br>
 +
I tried the same PCR with different annealing temperatures. The optimal annealing temperature is 62°C. I used a gradient between 58°C and 62°C. I again got no products.
 +
 
 +
<h3>09-15-2008</h3>
 +
'''CMV PCR'''<br>
 +
I again tried the CMV PCR, but this time with different polymerases. I used the Taq Polymerase and a Mix. I also tried one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc)
 +
I got products in the approach with taq polymerase as well as in the approach with the Mix. I cut out 3 bands from each approach.
 +
 
 +
<h3>09-16-2008</h3>
 +
'''Gel purification'''<br>
 +
I did a gel purification of the PCR products.
 +
 
 +
'''Digestion of the PCR products and the transfectionvector'''<br>
 +
Digestion with EcoRI and PstI. Then Kathrin did a ligation.
 +
 
 +
<h3>09-18-2008</h3>
 +
'''Transformation'''<br>
 +
I did a transformation with the ligation product.
 +
 
 +
<h3>09-19-2008</h3>
 +
'''Transformation'''<br>
 +
There were no colonies on the plates.
 +
 
 +
<h3>09-20-2008</h3>
 +
'''Digestion of the PCR products and the transfectionvector'''<br>
 +
Now I used the restrictionenzymes XbaI and SpeI.
 +
 
 +
'''Gel purification and ligation'''<br>
 +
The digested PCR products and the vector.
 +
 
 +
<h3>09-21-2008</h3>
 +
'''Transformation of the ligation'''<br>
 +
I used 10 µl of the ligation and used RV 308 cells
}}
}}

Revision as of 18:32, 9 October 2008


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__september



09-10-2008


CMV PCR
I did a PCR with primer for the CMV promotor. As DNA template I used the CMV+RLuc construct from the Ljubljana group which I isolated from the parts collection 2007.

For a 50 µl reaction I used:
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

I got no product.

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
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
I tried the same PCR with different annealing temperatures. The optimal annealing temperature is 62°C. I used a gradient between 58°C and 62°C. I again got no products.

09-15-2008

CMV PCR
I again tried the CMV PCR, but this time with different polymerases. I used the Taq Polymerase and a Mix. I also tried one approach with the complete plasmid and one with the digested plasmid (see digestion of CMV+Rluc) I got products in the approach with taq polymerase as well as in the approach with the Mix. I cut out 3 bands from each approach.

09-16-2008

Gel purification
I did a gel purification of the PCR products.

Digestion of the PCR products and the transfectionvector
Digestion with EcoRI and PstI. Then Kathrin did a ligation.

09-18-2008

Transformation
I did a transformation with the ligation product.

09-19-2008

Transformation
There were no colonies on the plates.

09-20-2008

Digestion of the PCR products and the transfectionvector
Now I used the restrictionenzymes XbaI and SpeI.

Gel purification and ligation
The digested PCR products and the vector.

09-21-2008

Transformation of the ligation
I used 10 µl of the ligation and used RV 308 cells

Freiburg08 FT3.png

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