Team:Marburg/Notebook:June

From 2013.igem.org

(Difference between revisions)
Line 151: Line 151:
</div>
</div>
</fieldset>
</fieldset>
-
</div>
+
 
<fieldset class="experiment ligation">
<fieldset class="experiment ligation">
     <legend><a name="lig">Ligation</a></legend>
     <legend><a name="lig">Ligation</a></legend>
Line 174: Line 174:
</div>
</div>
</fieldset>
</fieldset>
 +
</div>
<div class="notebooky-entry">
<div class="notebooky-entry">
Line 376: Line 377:
</div>
</div>
</fieldset>
</fieldset>
-
</div>
 
 +
<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">Digest of pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9, pSB1C3-iBB6315 and pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="digest">
 +
<li>1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)</li>
 +
<li>0.5 µl EcoRI</li>
 +
<li>0.5 µl SpeI</li>
 +
<li>2 µl CutSmart</li>
 +
<li>ad 20 µl ddH2O</li>
 +
</ul>
 +
 +
<p><ul class="digest">
 +
<li>1000 ng Plasmid (pSB1C3-iBB6315)</li>
 +
<li>0.5 µl XbaI</li>
 +
<li>0.5 µl PstI</li>
 +
<li>2 µl CutSmart</li>
 +
<li>ad 20 µl ddH2O</li>
 +
</ul>
 +
 +
<p><ul class="digest">
 +
<li>1000 ng Plasmid (pSB1A3)</li>
 +
<li>0.5 µl EcoRI</li>
 +
<li>0.5 µl PstI</li>
 +
<li>2 µl CutSmart</li>
 +
<li>ad 20 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="beispiel.png" width="50%" 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>x µl Hyper Ladder</li>
 +
</ul>
 +
</p>
 +
<p>
 +
<span class="exp">Expactations</span>
 +
<ul class="exp">
 +
<li>Lane 1: 5300 kbp</li>
 +
<li>Lane 2: 3300 kbp</li>
 +
<li>Lane 3: 1300 kbp</li>
 +
</ul>
 +
</p>
 +
<p>All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</fieldset>
 +
 +
<fieldset class="experiment ligation">
 +
    <legend><a name="lig">Ligation</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">Assemble the biobricks iBB4+iBB11+iBB96315 and iBB4+iBB12+iBB96315 into the vector pSB1A3.</span>
 +
</div>
 +
<div class="exp-content">
 +
<p><ul class="lig">
 +
<li>2 µl vector DNA (pSB1A3)</li>
 +
<li>5 µl insert 1 DNA (iBB6315)</li>
 +
<li>5 µl insert 2 DNA (iBB4+iBB11+iBB9, iBB4+iBB12+iBB9)</li>
 +
<li>2 µl 10x T4 DNA ligase buffer</li>
 +
<li>2 µl T4 DNA ligase</li>
 +
<li>4 µl ddH2O</li>
 +
</ul>
 +
<p>The samples were incubated for 2h at 16 °C.</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+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+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+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.<br />
 +
The plates were incubated over night at 37° C.</p>
 +
</div>
 +
</fieldset>
 +
</div>
</html>
</html>
{{:Team:Marburg/Template:ContentEnd}}
{{:Team:Marburg/Template:ContentEnd}}
{{:Team:Marburg/Template:Footer}}
{{:Team:Marburg/Template:Footer}}

Revision as of 13:20, 26 September 2013

Notebook

25.06.2013

Digest
Investigator: Dominik
Aim: Test digest of pSB1A3-iBB39, pSB1A3-iBB49 and pSB1C3-iBB6315 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB39,pSB1A3-iBB49 and pSB1C3-iBB6315)
  • 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
  • x µl Hyper Ladder

Expactations

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

Worked as expected.

26.06.2013

Digest
Investigator: Dominik
Aim: Construction of the plasmids pSB1A3-iBB10+9, pSB1A3-iBB11+9, pSB1A3-iBB12+9 and pSB1A3-iBB13+9

  • 1 µl Plasmid (pSB1C3-iBB10, pSB1C3-iBB11, pSB1C3-iBB12 and pSB1C3-iBB13)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • 12 µl ddH2O

  • 1 µl Plasmid (pSB1C3-iBB9)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • 12 µl ddH2O

  • 1 µl Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • 12 µl ddH2O

The samples were incubated for 1.5 h at 37° C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

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

Expactations

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

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator: Dominik
Aim: Assemble the biobricks iBB10+iBB9, iBB11+iBB9, iBB12+iBB9 and iBB13+iBB9 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 2 µl insert 1 DNA (iBB9)
  • 2 µl insert 2 DNA (iBB10, iBB11, iBB12 or iBB13)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 2 µl ddH2O

The samples were incubated overnight at room temperature.

27.06.2013

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9,pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

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

4 colonies of pSB1A3-iBB395146 and 4 colonies of pSB1A3-iBB496315 were inoculated in 5 ml LB medium containing ampicillin.

02.07.2013

Miniprep
Investigator: Dominik
Aim: preparation of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9.

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

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1A3-iBB10+iBB9, pSB1A3-iBB11+iBB9, pSB1A3-iBB12+iBB9 and pSB1A3-iBB13+iBB9)
  • 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
  • x µl Hyper Ladder

Expactations

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

Worked as expected.

23.07.2013

Digest
Investigator: Dominik
Aim: Test digest of the plasmids pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9 with EcoRI and PstI

  • 1 µl Plasmid (pSB1C3-iBB4+iBB11+iBB9 and pSB1C3-iBB4+iBB12+iBB9)
  • 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
  • x µl Hyper Ladder

Expactations

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

Worked as expected.

24.07.2013

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

4 colonies of pSB1C3-iBB4+iBB10+iBB9 and 4 colonies of pSB1C3-iBB4+iBB13+iBB9 were inoculated in 5 ml LB medium containing chloramphenicol.

Digest
Investigator: Dominik
Aim: Digest of pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9, pSB1C3-iBB6315 and pSB1A3.

  • 1000 ng Plasmid (pSB1C3-iBB4+iBB11+iBB9, pSB1C3-iBB4+iBB12+iBB9)
  • 0.5 µl EcoRI
  • 0.5 µl SpeI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1C3-iBB6315)
  • 0.5 µl XbaI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 µl ddH2O

  • 1000 ng Plasmid (pSB1A3)
  • 0.5 µl EcoRI
  • 0.5 µl PstI
  • 2 µl CutSmart
  • ad 20 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
  • x µl Hyper Ladder

Expactations

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

All expected fragments were present. The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf).
Ligation
Investigator: Dominik
Aim: Assemble the biobricks iBB4+iBB11+iBB96315 and iBB4+iBB12+iBB96315 into the vector pSB1A3.

  • 2 µl vector DNA (pSB1A3)
  • 5 µl insert 1 DNA (iBB6315)
  • 5 µl insert 2 DNA (iBB4+iBB11+iBB9, iBB4+iBB12+iBB9)
  • 2 µl 10x T4 DNA ligase buffer
  • 2 µl T4 DNA ligase
  • 4 µl ddH2O

The samples were incubated for 2h at 16 °C.

Transformation
Investigator: Dominik
Aim: Transformation of E. coli DH5α with the plasmid DNA pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315.

The chemo competent E. coli DH5α cells were transformed with the plasmids pSB1A3-iBB4+iBB11+iBB96315 and pSB1A3-iBB4+iBB12+iBB96315 DNA and plated on LB-Amp-plates.
The plates were incubated over night at 37° C.

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