Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

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</div>
</div>
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<div class="cyt_frag3_headline">
 
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<a name="cyt_frag3"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for mtrCAB Fragment3</span></a>
 
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</div>
 
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<div class="cyt_frag3">
 
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<table border=1 style="float:left; padding-left:10px">
 
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      <col width="150">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
 
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<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
 
-
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
 
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<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
 
-
<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
 
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<tr> <td>Phusion (0.5u)</td>        <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>H2O</td>          <td align="right">29.5</td> <td align="right">259.6</td> </tr>
 
-
 
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</table>
 
-
 
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<table border=1 style="float:left; margin-left:20px">
 
-
      <col width="50">
 
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      <col width="50">
 
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      <col width="50">
 
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      <col width="50">
 
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      <col width="50">
 
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<tr><th colspan="5">Program Phusion</th></tr>
 
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<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
 
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<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>50s</td><td>6:00</td></tr>
 
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<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 
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<tr><td colspan="5">
 
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<b><u>Notes:</u></b>
 
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<br>Size: 3000bp
 
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<br>Primer1:
 
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<br>Primer2:
 
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<br>
 
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</td></tr>
 
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</table>
 
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<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
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</div>
 
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<div class="cyt_psb1c3_headline">
 
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<a name="cyt_psb1c3"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for psB1C3 with and without Gibson-overlap for mtrCAB </span></a>
 
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</div>
 
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<div class="cyt_psb1c3">
 
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<table border=1 style="float:left; padding-left:10px">
 
-
      <col width="150">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
 
-
<tr> <td>GC-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
 
-
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
 
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<tr> <td>Primer1 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
 
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<tr> <td>Primer2 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
 
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<tr> <td>Template (5.5ng/ul)</td><td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>DMSO</td>         <td align="right">1.5</td>    <td align="right">13.2</td> </tr>
 
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<tr> <td>Phusion (0.5u)</td>        <td align="right">0.5</td> <td align="right">4.4</td> </tr>
 
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<tr> <td>H2O</td>          <td align="right">32.5</td> <td align="right">286.0</td> </tr>
 
-
 
-
</table>
 
-
 
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<table border=1 style="float:left; margin-left:20px">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
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<tr><th colspan="5">Program Phusion</th></tr>
 
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<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
 
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<tr align="center"><td>45s</td> <td>30s</td><td>1:00</td> <td>45s</td><td>5:00</td></tr>
 
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<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 
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<tr><td colspan="5">
 
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<b><u>Notes:</u></b>
 
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<br>Size: 2070bp
 
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<br>Primer1_Gibson:
 
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<br>Primer2_Gibson:
 
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<br>Primer1:
 
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<br>Primer2:
 
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<br>
 
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</td></tr>
 
-
 
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</table>
 
-
 
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<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
-
</div>
 
-
 
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<div class="cyt_mtrCAB_headline">
 
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<a name="cyt_mtrCAB"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for mtrCAB cluster </span></a>
 
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</div>
 
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<div class="cyt_mtrCAB">
 
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<table border=1 style="float:left; padding-left:10px">
 
-
      <col width="150">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
 
-
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
 
-
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.5</td> <td align="right">22.0</td> </tr>
 
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<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>Template (5.5ng/ul)</td><td align="right">2.0</td> <td align="right">17.6</td> </tr>
 
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<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
 
-
<tr> <td>Phusion (0.5u)</td>        <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
-
<tr> <td>H2O</td>          <td align="right">29.5</td> <td align="right">259.6</td> </tr>
 
-
 
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</table>
 
-
 
-
<table border=1 style="float:left; margin-left:20px">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
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<tr><th colspan="5">Program Phusion</th></tr>
 
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<tr align="center"><td>98 °C</td><td>98 °C</td><td>60 °C</td><td>72 °C</td><td>72 °C</td></tr>
 
-
<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>1:15</td><td>6:15</td></tr>
 
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<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 
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<tr><td colspan="5">
 
-
<b><u>Notes:</u></b>
 
-
<br>Size: 5150bp
 
-
<br>Primer1:
 
-
<br>Primer2:
 
-
<br>
 
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</td></tr>
 
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</table>
 
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<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
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</div>
 
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<div class="cyt_ccmAH_headline">
 
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<a name="cyt_ccmAH"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for ccmAH cluster</span></a>
 
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</div>
 
-
<div class="cyt_ccmAH">
 
-
<table border=1 style="float:left; padding-left:10px">
 
-
      <col width="150">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
<tr> <th>Reagent</th>         <th>1x</th> <th>8x</th> </tr>
 
-
<tr> <td>Hf-Buffer 5x</td>      <td align="right">10.0</td> <td align="right">88.0</td> </tr>
 
-
<tr> <td>dNTPs (10mM)</td>  <td align="right">2.0</td> <td align="right">17.6</td> </tr>
 
-
<tr> <td>Primer1 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
-
<tr> <td>Primer2 (10mM)</td> <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>Template (5.5ng/ul)</td><td align="right">3.0</td> <td align="right">26.4</td> </tr>
 
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<tr> <td>DMSO</td>         <td align="right">3.0</td>    <td align="right">26.4</td> </tr>
 
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<tr> <td>Phusion (0.5u)</td>        <td align="right">1.0</td> <td align="right">8.8</td> </tr>
 
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<tr> <td>H2O</td>          <td align="right">29.5</td> <td align="right">255.2</td> </tr>
 
-
 
-
</table>
 
-
 
-
<table border=1 style="float:left; margin-left:20px">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
-
      <col width="50">
 
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<tr><th colspan="5">Program Phusion</th></tr>
 
-
<tr align="center"><td>98 °C</td><td>98 °C</td><td>68 °C</td><td>72 °C</td><td>72 °C</td></tr>
 
-
<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>1:35</td><td>6:00</td></tr>
 
-
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 
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<tr><td colspan="5">
 
-
<b><u>Notes:</u></b>
 
-
<br>Size: 6311bp
 
-
<br>Primer1:
 
-
<br>Primer2:
 
-
<br>
 
-
</td></tr>
 
-
 
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</table>
 
-
 
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<br><br><br><br><br><br><br><br><br><br><br><br><br>
 
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</div>
 

Revision as of 21:17, 25 September 2013







Genetic enginering protocols


Standard Protocols


[Standard Protocols] Standard protocols
Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.0 17.6
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (16.5ng) 3.0 26.4
DMSO 4.0 35.2
Phusion (0.5u) 1.0 8.8
H2O 26.0 228.8













Specific Protocols


[Cytochromes] Phusion 50ul for mtrCAB Fragment1
Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.0 17.6
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (16.5ng) 3.0 26.4
DMSO 4.0 35.2
Phusion (0.5u) 1.0 8.8
H2O 26.0 228.8
Program Phusion
98 °C98 °C68 °C72 °C72 °C
30s 10s30s 1:356:00
35 cycles
Notes:
Size: 1800bp
Primer1:
Primer2:












[Cytochromes] Phusion 50ul for mtrCAB Fragment2
Reagent 1x 8x
Hf-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.5 22.0
Primer1 (10mM) 1.0 8.8
Primer2 (10mM) 1.0 8.8
Template (5.5ng/ul)2.0 17.6
DMSO 3.0 26.4
Phusion (0.5u) 1.0 8.8
H2O 29.5 259.6
Program Phusion
98 °C98 °C60 °C72 °C72 °C
1:00 10s30s 30s6:00
35 cycles
Notes:
Size: 330bp
Primer1:
Primer2:







































Retrieved from "http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/Molecular"