Team:Marburg/Notebook:August

From 2013.igem.org

(Difference between revisions)
Line 1: Line 1:
{{:Team:Marburg/Template:Header}}
{{:Team:Marburg/Template:Header}}
 +
{{:Team:Marburg/Template:ContentTitleNav}}
 +
Notebook: August
 +
{{:Team:Marburg/Template:ContentStartNav}}
 +
 +
<html>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="01-08-2013">01.08.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment sequencing">
 +
    <legend>Sequencing</le{{:Team:Marburg/Template:Header}}
{{:Team:Marburg/Template:ContentTitleNav}}
{{:Team:Marburg/Template:ContentTitleNav}}
Notebook: August
Notebook: August
Line 13: Line 26:
<fieldset class="experiment sequencing">
<fieldset class="experiment sequencing">
     <legend>Sequencing</legend>
     <legend>Sequencing</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>2 additional sequence samples were sent out for sequencing.</p>
 +
</div>
 +
</fieldset>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Test digest of pSB1A3-iBB4+iBB13+iBB96315 with <i>Eco</i>RI and <i>Pst</i>I</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)</li>
 +
<li>0.3 µl <i>Eco</i>RI</li>
 +
<li>0.3 µl <i>Pst</I></li>
 +
<li>1 µl CutSmart</li>
 +
<li>7.4 µl ddH2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1 h at 37° C.</p>
 +
<p>After that, a gel electrophoresis was made, but failed and has to be repeated the next day</p>
 +
</div>
 +
</fieldset>
 +
 +
<fieldset class="experiment inoculation">
 +
    <legend><a name="ino">Inoculation</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">inoculation of colonies for plasmid preparation</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.</p>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="02-08-2013">02.08.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment sequencing">
 +
    <legend>Sequencing</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Analysis of the sequence of pSB1A3-iBB496315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.</p>
 +
</div>
 +
</fieldset>
 +
 +
<fieldset class="experiment transformation">
 +
    <legend>Transformation</legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Transformation of <i>E. coli</i> DH5&#945; with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>The chemo competent <i>E. coli</i> DH5&alpha; cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.<br />
 +
The plates were incubated over night at 37° C.</p>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="03-08-2013">03.08.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment miniprep">
 +
    <legend><a name="min">Miniprep</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">preparation of the plasmid pSB1A3-iBB496315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p>The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.</p>
 +
</div>
 +
</fieldset>
 +
 +
<fieldset class="experiment digest">
 +
    <legend><a name="dig">Digest</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with <i>Eco</i>RI and <i>Pst</i>I</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="digest">
 +
<li>1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)</li>
 +
<li>0.3 µl <i>EcoR</i>I</li>
 +
<li>0.3 µl <i>PstI</i></li>
 +
<li>1 µl CutSmart</li>
 +
<li>7.4 µl ddH2O</li>
 +
</ul>
 +
<p>The samples were incubated for 1 h at 37° C.</p>
 +
<table class="gel digest">
 +
<colgroup>
 +
<col width="50%" />
 +
<col width="50%" />
 +
</colgroup>
 +
<thead>
 +
<tr>
 +
<th colspan="2" class="title">Gel electrophoresis</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<img src="https://static.igem.org/mediawiki/2013/2/26/Gel_2013-08-02.png" width="60%" alt="gel-electrophoresis-image" />
 +
</td>
 +
<td>
 +
<p>
 +
<span class="gel-elc">Gel substances</span>
 +
<ul class="gel-sub">
 +
<li>1% Agarose gel</li>
 +
<li>10 µl RedSafe in 50 ml gel</li>
 +
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
 +
</ul>
 +
<p>Worked as expected.</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
<div class="notebooky-entry">
 +
<h2 class="title">
 +
<a name="14-08-2013">14.08.2013</a>
 +
</h2>
 +
 +
<fieldset class="experiment sequencing">
 +
    <legend><a name="seq">Sequencing</a></legend>
 +
    <div class="investigator">
 +
<span class="inv">Investigator:</span>
 +
<span class="inv-names">Dominik</span>
 +
</div>
 +
    <div class="aim">
 +
<span class="aim">Aim:</span>
 +
<span class="aim-desc">Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="seq">
 +
<li>pSB1A3-iBB496315: Okay</li>
 +
<li>pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp &rarr; Leu. Will be ignored</li>
 +
<li>pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.</li>
 +
</ul>
 +
<p>All plasmids are brought to Marian who will transform them into <i>P. tricornutum</i> cells.</p>
 +
</div>
 +
</fieldset>
 +
</div>
 +
 +
</html>
 +
 +
{{:Team:Marburg/Template:ContentEndNav}}
 +
{{:Team:Marburg/Template:Footer}}
 +
gend>
     <div class="investigator">
     <div class="investigator">
<span class="inv">Investigator:</span>
<span class="inv">Investigator:</span>

Revision as of 21:43, 3 October 2013

Notebook: August

01.08.2013

Sequencing

01.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

2 additional sequence samples were sent out for sequencing.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
  • 0.3 µl EcoRI
  • 0.3 µl Pst
  • 1 µl CutSmart
  • 7.4 µl ddH2O

The samples were incubated for 1 h at 37° C.

After that, a gel electrophoresis was made, but failed and has to be repeated the next day

Inoculation
Investigator: Dominik
Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

02.08.2013

Sequencing
Investigator: Dominik
Aim: Analysis of the sequence of pSB1A3-iBB496315.

Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

03.08.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmid pSB1A3-iBB496315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O

The samples were incubated for 1 h at 37° C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)

Worked as expected.

14.08.2013

Sequencing
Investigator: Dominik
Aim: Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

  • pSB1A3-iBB496315: Okay
  • pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp → Leu. Will be ignored
  • pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.

All plasmids are brought to Marian who will transform them into P. tricornutum cells.

gend>

Investigator: Dominik

Aim: Examination of the sequence of pSB1A3-iBB4+iBB10+iBB96315.

2 additional sequence samples were sent out for sequencing.

</fieldset>

<fieldset class="experiment digest"> 

   <legend><a name="dig">Digest</a></legend>

Investigator: Dominik

Aim: Test digest of pSB1A3-iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
  • 0.3 µl EcoRI
  • 0.3 µl Pst
  • 1 µl CutSmart
  • 7.4 µl ddH2O
<p>The samples were incubated for 1 h at 37° C.

After that, a gel electrophoresis was made, but failed and has to be repeated the next day

</fieldset>

<fieldset class="experiment inoculation"> 

   <legend><a name="ino">Inoculation</a></legend>

Investigator: Dominik

Aim: inoculation of colonies for plasmid preparation

4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

</fieldset> </div>

<a name="02-08-2013">02.08.2013</a>

<fieldset class="experiment sequencing"> 

   <legend>Sequencing</legend>

Investigator: Dominik

Aim: Analysis of the sequence of pSB1A3-iBB496315.

Obviously the wrong plasmid was sent out for sequencing. Due to the lack of enough plasmids for an additional sequence analysis, we will redo a new miniprep.

</fieldset>

<fieldset class="experiment transformation"> 

   <legend>Transformation</legend>

Investigator: Dominik

Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315 and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

</fieldset>

<a name="03-08-2013">03.08.2013</a>

<fieldset class="experiment miniprep"> 

   <legend><a name="min">Miniprep</a></legend>

Investigator: Dominik

Aim: preparation of the plasmid pSB1A3-iBB496315.

The plasmids were isolated with the “QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf) according to the producer instructions.

</fieldset>

<fieldset class="experiment digest"> 

   <legend><a name="dig">Digest</a></legend>

Investigator: Dominik

Aim: Test digest of pSB1A3-iBB496315 and iBB4+iBB13+iBB96315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB496315 and iBB4+iBB13+iBB96315)
  • 0.3 µl EcoRI
  • 0.3 µl PstI
  • 1 µl CutSmart
  • 7.4 µl ddH2O
<p>The samples were incubated for 1 h at 37° C.

<colgroup> <col width="50%" /> <col width="50%" /> </colgroup> <thead> </thead> <tbody> </tbody>
Gel electrophoresis

<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • x µl Hyper Ladder

Expectations

  • Lane 1: 5300 kbp
  • Lane 2: 3300 kbp
  • Lane 3: 1300 kbp

Worked as expected.

</fieldset>

<a name="14-08-2013">14.08.2013</a>

<fieldset class="experiment sequencing"> 

   <legend><a name="seq">Sequencing</a></legend>

Investigator: Dominik

Aim: Examination of sequence analysis of the plasmids pSB1A3-iBB496315, pSB1A3-iBB4+iBB10+iBB96315 and pSB1A3-iBB4+iBB13+iBB96315.

  • pSB1A3-iBB496315: Okay
  • pSB1A3-iBB4+iBB10+iBB96315: mutation in signal peptide that leads to Trp → Leu. Will be ignored
  • pSB1A3-iBB4+iBB13+iBB96315: mutation in terminator, will be ignored.
<p>All plasmids are brought to Marian who will transform them in P. tricornutum cells.

</fieldset>

</html>

Retrieved from "http://2013.igem.org/Team:Marburg/Notebook:August"