Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

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.standard_protocol{display:none;}
.cyt_frag1{display:none;}
.cyt_frag1{display:none;}
.cyt_frag2{display:none;}
.cyt_frag2{display:none;}
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.cyt_frag3{display:none;}
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.cyt_psb1c3{display:none;}
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.cyt_mtrCAB{display:none;}
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.cyt_ccmAH{display:none;}
.h1{color:#ff6600;}
.h1{color:#ff6600;}
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<div id=globalwrapper style="padding-left:20px; padding-right:20px;">
 
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<br><br>
 
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<h1 style="color:#ff6600;"> Genetic enginering protocols</h1>
 
-
<br>
 
-
<!--<h2>Standard Protocols</h2> -->
 
-
<div class="iso_whole_headline">
 
-
<a name="iso_whole"><span style="color:#ff6600">[Whole Genome Isolation]</span></a>
 
 +
<div id=globalwrapper style="padding-left:20px; padding-right:20px;">
-
</div>
+
<br><br><br>
-
<div class="iso_whole">
+
<h1>PCR Protocols and Programs</h1>
<br>
<br>
-
For the isolation following kit has been used:
+
<h2>Standard Protocols</h2>
-
Fermentas GeneJET™ Plasmid Miniprep Kit
+
-
 
+
-
<p> - Centrifuge 10 mL of over-night liquid culture</p>
+
-
<p> - Resuspend pellet in 800 µL of resuspension solution</p>
+
-
<p> - Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)</p>
+
-
<p> - Centrifuge 3 min at 10,000 rpm</p>
+
-
<p> - Transfer 500 µL of supernatant into new 2 mL tube</p>
+
-
<p> - Add 500 µL of lysis buffer, invert 4 - 6 times</p>
+
-
<p> - Add 700 µL of neutralisation buffer, invert 4 - 6 times</p>
+
-
<p> - Centrifuge 10 min at 12,000 rpm</p>
+
-
<p> - Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)</p>
+
-
<p> - Two wash steps with 750 µL of washing buffer</p>
+
-
<p> - Dry column</p>
+
-
<p> - Elute in 75 µL of elution buffer</p>
+
-
<p> -</p>
+
-
<p> -</p>
+
-
<p> -</p>
+
-
<p> -</p>
+
<br>
<br>
 +
<div class="standard_headline">
 +
<a name="standard_protocol"><span style="color:#ff6600">[Standard Protocols]</span> Standard protocols</span></a>
 +
</div>
 +
<div class="standard_protocol">
 +
<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>
 +
<tr> <td>Template (16.5ng)</td> <td align="right">3.0</td> <td align="right">26.4</td> </tr>
 +
<tr> <td>DMSO</td>         <td align="right">4.0</td>    <td align="right">35.2</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">26.0</td> <td align="right">228.8</td> </tr>
 +
</table>
-
<!--
+
<br><br><br><br><br><br><br><br><br><br><br>
-
<p> -</p>
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<p> -</p>
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<br>
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</div>
</div>
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</div>
</div>
 +
 +
<div class="cyt_frag3_headline">
 +
<a name="cyt_frag3"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for mtrCAB Fragment3</span></a>
 +
</div>
 +
<div class="cyt_frag3">
 +
<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>
 +
<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>
 +
<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>
 +
<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>
 +
 +
</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">
 +
<tr><th colspan="5">Program Phusion</th></tr>
 +
<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
 +
<tr align="center"><td>30s</td> <td>10s</td><td>30s</td> <td>50s</td><td>6:00</td></tr>
 +
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 +
<tr><td colspan="5">
 +
<b><u>Notes:</u></b>
 +
<br>Size: 3000bp
 +
<br>Primer1:
 +
<br>Primer2:
 +
<br>
 +
</td></tr>
 +
 +
</table>
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
</div>
 +
 +
<div class="cyt_psb1c3_headline">
 +
<a name="cyt_psb1c3"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for psB1C3 with and without Gibson-overlap for mtrCAB </span></a>
 +
</div>
 +
<div class="cyt_psb1c3">
 +
<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>
 +
<tr> <td>Primer1 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
 +
<tr> <td>Primer2 (10mM)</td> <td align="right">1.25</td> <td align="right">11.0</td> </tr>
 +
<tr> <td>Template (5.5ng/ul)</td><td align="right">1.0</td> <td align="right">8.8</td> </tr>
 +
<tr> <td>DMSO</td>         <td align="right">1.5</td>    <td align="right">13.2</td> </tr>
 +
<tr> <td>Phusion (0.5u)</td>        <td align="right">0.5</td> <td align="right">4.4</td> </tr>
 +
<tr> <td>H2O</td>          <td align="right">32.5</td> <td align="right">286.0</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">
 +
<tr><th colspan="5">Program Phusion</th></tr>
 +
<tr align="center"><td>98 °C</td><td>98 °C</td><td>70 °C</td><td>72 °C</td><td>72 °C</td></tr>
 +
<tr align="center"><td>45s</td> <td>30s</td><td>1:00</td> <td>45s</td><td>5:00</td></tr>
 +
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 +
<tr><td colspan="5">
 +
<b><u>Notes:</u></b>
 +
<br>Size: 2070bp
 +
<br>Primer1_Gibson:
 +
<br>Primer2_Gibson:
 +
<br>Primer1:
 +
<br>Primer2:
 +
<br>
 +
</td></tr>
 +
 +
</table>
 +
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
</div>
 +
 +
<div class="cyt_mtrCAB_headline">
 +
<a name="cyt_mtrCAB"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for mtrCAB cluster </span></a>
 +
</div>
 +
<div class="cyt_mtrCAB">
 +
<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>
 +
<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>
 +
<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>
 +
<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>
 +
 +
</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">
 +
<tr><th colspan="5">Program Phusion</th></tr>
 +
<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>
 +
<tr><td></td><td colspan="3" align="center">35 cycles</td><td></td></tr>
 +
<tr><td colspan="5">
 +
<b><u>Notes:</u></b>
 +
<br>Size: 5150bp
 +
<br>Primer1:
 +
<br>Primer2:
 +
<br>
 +
</td></tr>
 +
 +
</table>
 +
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
</div>
 +
 +
<div class="cyt_ccmAH_headline">
 +
<a name="cyt_ccmAH"><span style="color:#ff6600">[Cytochromes]</span> Phusion 50ul for ccmAH cluster</span></a>
 +
</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>
 +
<tr> <td>Template (5.5ng/ul)</td><td align="right">3.0</td> <td align="right">26.4</td> </tr>
 +
<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">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">
 +
<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>
 +
<tr><td colspan="5">
 +
<b><u>Notes:</u></b>
 +
<br>Size: 6311bp
 +
<br>Primer1:
 +
<br>Primer2:
 +
<br>
 +
</td></tr>
 +
 +
</table>
 +
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
</div>

Revision as of 21:33, 25 September 2013







PCR Protocols and Programs


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:













[Cytochromes] Phusion 50ul for mtrCAB Fragment3
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 °C70 °C72 °C72 °C
30s 10s30s 50s6:00
35 cycles
Notes:
Size: 3000bp
Primer1:
Primer2:













[Cytochromes] Phusion 50ul for psB1C3 with and without Gibson-overlap for mtrCAB
Reagent 1x 8x
GC-Buffer 5x 10.0 88.0
dNTPs (10mM) 2.0 17.6
Primer1 (10mM) 1.25 11.0
Primer2 (10mM) 1.25 11.0
Template (5.5ng/ul)1.0 8.8
DMSO 1.5 13.2
Phusion (0.5u) 0.5 4.4
H2O 32.5 286.0
Program Phusion
98 °C98 °C70 °C72 °C72 °C
45s 30s1:00 45s5:00
35 cycles
Notes:
Size: 2070bp
Primer1_Gibson:
Primer2_Gibson:
Primer1:
Primer2:













[Cytochromes] Phusion 50ul for mtrCAB cluster
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
30s 10s30s 1:156:15
35 cycles
Notes:
Size: 5150bp
Primer1:
Primer2:













[Cytochromes] Phusion 50ul for ccmAH cluster
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 (5.5ng/ul)3.0 26.4
DMSO 3.0 26.4
Phusion (0.5u) 1.0 8.8
H2O 29.5 255.2
Program Phusion
98 °C98 °C68 °C72 °C72 °C
30s 10s30s 1:356:00
35 cycles
Notes:
Size: 6311bp
Primer1:
Primer2:







































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