Team:Marburg/Notebook:March

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Notebook: March
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     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Preparation of BioBrick-templates &rarr; Transformation into <i>E. coli</i> DH5&alpha;.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Transformation into <i>E. coli</i> DH5&alpha;.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with H2O) in <i>E. coli</i> DH5&alpha; (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with bidest. H2O) in <i>E. coli</i> DH5&alpha; (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 45: Line 43:
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Preparation of BioBrick-templates &rarr; Inoculation of transformants for miniprep.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Inoculation of transformants for miniprep.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LB<sub>amp</sub>, oN 37°C.</p>
+
<p>2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37°C.</p>
-
<p>pCAT is a Cosmid, transformation failed.</p>
+
<p>pCAT is a cosmid, transformation failed.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 70: Line 68:
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Preparation of BioBrick-templates &rarr; Miniprep of the template-plasmids.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Miniprep of the template-plasmids.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 25 µl H2O.</p>
+
<p>Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 86: Line 84:
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Preparation of BioBrick-templates &rarr; Control digestion of the template plasmids.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Control digestion of the template plasmids.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
Line 94: Line 92:
<li>1 µl enzyme 2</li>
<li>1 µl enzyme 2</li>
<li>4 µl buffer 2</li>
<li>4 µl buffer 2</li>
-
<li>31 µl H2O</li>
+
<li>31 µl bidest. H2O</li>
</ul>
</ul>
<p>Samples:</p>
<p>Samples:</p>
<p>Digestion of pPha-NR with <i>EcoR</i>I and <i>Nco</i>I.</p>
<p>Digestion of pPha-NR with <i>EcoR</i>I and <i>Nco</i>I.</p>
<p>Digestion of pPha-T1 with <i>EcoR</i>I and <i>Nde</i>I.</p>
<p>Digestion of pPha-T1 with <i>EcoR</i>I and <i>Nde</i>I.</p>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 196: Line 194:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Phusion Polymerase</td>
+
<td>Phusion polymerase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 203: Line 201:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Primer fw (100pmol/µl)</td>
+
<td>Primer fw (100 pmol/µl)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>95</td>
<td>95</td>
Line 210: Line 208:
<tr>
<tr>
<td>1 µl</td>
<td>1 µl</td>
-
<td>Primer rv (100pmol/µl) </td>
+
<td>Primer rv (100 pmol/µl) </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>65</td>
<td>65</td>
Line 239: Line 237:
<tr>
<tr>
<td>35 µl</td>
<td>35 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
Line 365: Line 363:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Transformation of 1 µl PCR-product into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, oN 37°C.</p>
+
<p>Transformation of 1 µl PCR-product into <i>E. coli</i> DH5&alpha; (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
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<div class="exp-content">
<div class="exp-content">
<p>Transformation of iBB5 failed, new mutagenesis primers ordered.</p>
<p>Transformation of iBB5 failed, new mutagenesis primers ordered.</p>
-
<p>3 colonies of iBB3 and iBB4 inoculated in 5 ml LB<sub>amp</sub>, oN 37°C.</p>
+
<p>3 colonies of iBB3 and iBB4 inoculated in 5 ml LB<sub>amp</sub>, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 406: Line 404:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Inoculation of Ca-competent <i>E. coli</i> DH5&alpha; in 2x5 ml LB, oN 37°C.</p>
+
<p>Inoculation of Ca-competent <i>E. coli</i> DH5&alpha; in 2x5 ml LB, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 419: Line 417:
     <div class="aim">
     <div class="aim">
<span class="aim">Aim:</span>
<span class="aim">Aim:</span>
-
<span class="aim-desc">Preparation of BioBrick-templates &rarr; Inoculation for glycerol stocks.</span>
+
<span class="aim-desc">Preparation of BioBrick templates &rarr; Inoculation for glycerol stocks.</span>
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>PPha-NR and pPha-T1 were inoculated in 5 ml LB, oN 37°C (for glycerol stocks).</p>
+
<p>PPha-NR and pPha-T1 were inoculated in 5 ml LB, overnight at 37°C (for glycerol stocks).</p>
</div>
</div>
</fieldset>
</fieldset>
Line 446: Line 444:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Miniprep of of iBB3 and iBB4 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.</p>
+
<p>Miniprep of of iBB3 and iBB4 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 540: Line 538:
<tr>
<tr>
<td>36 µl</td>
<td>36 µl</td>
-
<td>ddH20</td>
+
<td>bidest. H2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tr>
</table>
</table>
-
<br />
+
<p>The PCR was cleaned via 1% agarose gel: Relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.</p>
-
<!-- Gel-Bild-->
+
-
<table class="gel pcr">
+
-
<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>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
+
-
<li>6x loading buffer (for samples)</li>
+
-
</ul>
+
-
</p>
+
-
<p>The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.</p>
+
-
</td>
+
-
</tr>
+
-
</tbody>
+
-
</table>
+
</div>
</div>
</fieldset>
</fieldset>
Line 591: Line 558:
</div>
</div>
<div class="exp-content">
<div class="exp-content">
-
<p>Competent cells harvested and frozen.</p>
+
<p>Competent cells are harvested and frozen.</p>
<p>Controlled with several Transformations:</p>
<p>Controlled with several Transformations:</p>
<ul class="justalist">
<ul class="justalist">
Line 599: Line 566:
<li>cells from AG Bange as control</li>
<li>cells from AG Bange as control</li>
</ul>
</ul>
-
<p>30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, oN 37°C.</p>
+
<p>30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, overnight at 37°C.</p>
</div>
</div>
</fieldset>
</fieldset>
Line 620: Line 587:
<li>1 µl enzyme 2</li>
<li>1 µl enzyme 2</li>
<li>1 µl buffer</li>
<li>1 µl buffer</li>
-
<li>5 µl H2O</li>
+
<li>5 µl bidest. H2O</li>
</ul>
</ul>
<p>Samples:</p>
<p>Samples:</p>
-
<p>Digestion of iBB3 with <i>Pvu</i>II.</p>
+
<p>Digestion of iBB3 (P<sub>fcpB</sub>) with <i>Pvu</i>II.</p>
-
<p>Digestion of IBB4 with <i>Pst</i>I.</p>
+
<p>Digestion of IBB4 (P<sub>NR</sub>) with <i>Pst</i>I.</p>
-
<p>The samples were incubated for 1 h at 37° C.</p>
+
<p>The samples were incubated for 1 h at 37 °C.</p>
 +
<!-- Gel-Bild-->
<table class="gel digest">
<table class="gel digest">
<colgroup>
<colgroup>
Line 639: Line 607:
<tr>
<tr>
<td>
<td>
-
<img src="beispiel.png" width="50%" alt="gel-electrophoresis-image" />
+
<img src="https://static.igem.org/mediawiki/2013/3/3d/Gel_2013-03-27.png" width="100%" alt="gel-electrophoresis-image" />
</td>
</td>
<td>
<td>
Line 646: Line 614:
<ul class="gel-sub">
<ul class="gel-sub">
<li>1% Agarose gel</li>
<li>1% Agarose gel</li>
-
<li>10 µl RedSafe per 50 ml gel</li>
+
<li>10 µl RedSafe in 50 ml gel</li>
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
<li>6 µl GeneRuler&trade; 1kb plus DNA Ladder (Thermo Scientific)</li>
<li>6x loading buffer (for samples)</li>
<li>6x loading buffer (for samples)</li>
Line 652: Line 620:
</p>
</p>
<p>
<p>
-
<span class="exp">Expectations</span>
+
<table class="gel">
-
<ul class="exp">
+
<colgroup>
-
<li>iBB3: no cut, vector in different forms</li>
+
<col width="10%" />
-
<li>iBB4: 1 cut, linearized plasmid</li>
+
<col width="2%" />
-
</ul>
+
<col width="50%" />
 +
<col width="5%" />
 +
<col width="33%" />
 +
</colgroup>
 +
<thead>
 +
<th>Lane</th>
 +
<th>&nbsp;</th>
 +
<th>Content</th>
 +
<th>&nbsp;</th>
 +
<th>Comment</th>
 +
</thead>
 +
<tr>
 +
<td>2</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 1</td>
 +
<th>&nbsp;</th>
 +
<td rowspan="3">no cut, vector in different forms</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 2</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>fcpB</sub> 3</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>5</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 1</td>
 +
<th>&nbsp;</th>
 +
<td rowspan="3">1 cut &rarr; linearized plasmid</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 2</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<th>&nbsp;</th>
 +
<td>P<sub>NR</sub> 3</td>
 +
<th>&nbsp;</th>
 +
</tr>
 +
</table>
</p>
</p>
-
<p>All positive.</p>
 
</td>
</td>
</tr>
</tr>

Latest revision as of 11:12, 27 October 2013

Notebook: March Next Previous

20.03.2013

Transformation
Investigator: Christian
Aim: Preparation of BioBrick templates → Transformation into E. coli DH5α.

Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with bidest. H2O) in E. coli DH5α (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

21.03.2013

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation of transformants for miniprep.

2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LBamp, overnight at 37°C.

pCAT is a cosmid, transformation failed.

22.03.2013

Miniprep
Investigator: Christian
Aim: Preparation of BioBrick templates → Miniprep of the template-plasmids.

Miniprep of the liquid cultures (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 25 µl bidest. H2O.

Digest
Investigator: Christian
Aim: Preparation of BioBrick templates → Control digestion of the template plasmids.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 4 µl buffer 2
  • 31 µl bidest. H2O

Samples:

Digestion of pPha-NR with EcoRI and NcoI.

Digestion of pPha-T1 with EcoRI and NdeI.

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

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   pPha-NR   2868 bp + 992 bp
4+5   pPha-T1   3554 bp + 541 bp

All positive.
PCR
Investigator: Christian
Aim: Construction of BioBricks → Deletion of restriction sites in iBB3, iBB4 and iBB5 via mutagenesis PCR.

Mutagenesis PCR of PfcpB (iBB3), PNR (iBB4), and TfcpA (iBB5).

Volume Reagent   Temp (°C) Time
1 µl Phusion polymerase   95 3 min
1 µl Primer fw (100 pmol/µl)   95 30 sec
1 µl Primer rv (100 pmol/µl)   65 45 sec x19
1 µl Template pPha-NR (diluted 1:100)   72 4:10 min
1 µl dNTPs   72 6 min
5 µl 10x buffer   4 Hold
35 µl bidest. H2O  

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)
  • 6x loading buffer (for samples)

Lane   Content    
2   PfcpB    
3   PNR    
4   PfcpA    
5   PfcpB    
6   PNR    
7   PfcpA    

Digest
Investigator: Christian
Aim: Construction of BioBricks → Digestion of the template plasmid.

Digestion of the template with 1 µl DppI.

Transformation
Investigator: Christian
Aim: Construction of BioBricks → Transformation of mutagenised plasmids.

Transformation of 1 µl PCR-product into E. coli DH5α (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LBamp, overnight at 37°C.

26.03.2013

Inoculation
Investigator: Christian
Aim: Construction of BioBricks → Inoculation of transformants for miniprep.

Transformation of iBB5 failed, new mutagenesis primers ordered.

3 colonies of iBB3 and iBB4 inoculated in 5 ml LBamp, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

Inoculation of Ca-competent E. coli DH5α in 2x5 ml LB, overnight at 37°C.

Inoculation
Investigator: Christian
Aim: Preparation of BioBrick templates → Inoculation for glycerol stocks.

PPha-NR and pPha-T1 were inoculated in 5 ml LB, overnight at 37°C (for glycerol stocks).

27.03.2013

Miniprep
Investigator: Christian
Aim: Construction of BioBricks → Miniprep of transformants.

Miniprep of of iBB3 and iBB4 (QIAprep Spin Miniprep Kit (Qiagen)), eluted in 30 µl bidest. H2O.

Inoculation
Investigator: Christian
Aim: Production of chemo-competent E. coli cells → Inoculation of E. coli DH5α pre-culture.

3 ml E. coli DH5α pre-culture inoculated in 100 ml LB.

PCR
Investigator: Christian
Aim: Construction of BioBricks → Amplification of iBB2 via PCR.

PCR on CAT-cassette (iBB2):

Volume Reagent   Temp (°C) Time
1 µl Q5 Polymerase   95 3 min
1 µl Primer fw (1pmol/µl)   95 30 sec
1 µl Primer rv (1pmol/µl)   65 30 sec x26
1 µl Template   72 1 min
1 µl dNTPs   72 7 min
10 µl 5x Q5-buffer   4 Hold
36 µl bidest. H2O  

The PCR was cleaned via 1% agarose gel: Relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Transformation
Investigator: Christian
Aim: Production of chemo-competent E. coli → Control of cells.

Competent cells are harvested and frozen.

Controlled with several Transformations:

  • on LB
  • on LBamp
  • on LBamp with 1 µl pPha-T1
  • cells from AG Bange as control

30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, overnight at 37°C.

Digest
Investigator: Christian
Aim: Construction of BioBricks → Control digestion of mutations.
  • 3 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 1 µl buffer
  • 5 µl bidest. H2O

Samples:

Digestion of iBB3 (PfcpB) with PvuII.

Digestion of IBB4 (PNR) with PstI.

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)
  • 6x loading buffer (for samples)

Lane   Content   Comment
2   PfcpB 1   no cut, vector in different forms
3   PfcpB 2  
4   PfcpB 3  
5   PNR 1   1 cut → linearized plasmid
6   PNR 2  
7   PNR 3  

28.03.2013

Sequencing
Investigator: Christian
Aim: Construction of BioBricks → Sequencing.

IBB3 and iBB4 with point mutations prepared for sequencing:

  • AGBOOOK 128: iBB4 1
  • AGBOOOK 129: iBB4 2
  • AGBOOOK 130: iBB4 3
  • AGBOOOK 131: iBB3 1
  • AGBOOOK 132: iBB3 2
  • AGBOOOK 133: iBB3 3

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