Team:Bielefeld-Germany/Labjournal/Molecular

From 2013.igem.org

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<h1 style="color:#ff6600;"> Genetic enginering protocols</h1>
<h1 style="color:#ff6600;"> Genetic enginering protocols</h1>
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<h2>Standard Protocols</h2>
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<a name="iso_whole"><span style="color:#ff6600">[Whole Genome Isolation]</span></a>
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<a name="standard_protocol"><span style="color:#ff6600">[Standard Protocols]</span> Standard protocols</span></a>
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For the isolation following kit has been used:
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Fermentas GeneJET™ Plasmid Miniprep Kit
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<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>
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<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">1.0</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 (16.5ng)</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">4.0</td>    <td align="right">35.2</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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<p> - Centrifuge 10 mL of over-night liquid culture</p>
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<p> - Resuspend pellet in 800 µL of resuspension solution</p>
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<p> - Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)</p>
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<p> - Centrifuge 3 min at 10,000 rpm</p>
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<p> - Transfer 500 µL of supernatant into new 2 mL tube</p>
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<p> - Add 500 µL of lysis buffer, invert 4 - 6 times</p>
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<p> - Add 700 µL of neutralisation buffer, invert 4 - 6 times</p>
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<p> - Centrifuge 10 min at 12,000 rpm</p>
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<p> - Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)</p>
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<p> - Two wash steps with 750 µL of washing buffer</p>
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<p> - Dry column</p>
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<p> - Elute in 75 µL of elution buffer</p>
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Revision as of 21:28, 25 September 2013






Genetic enginering protocols


[Whole Genome Isolation]

For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit

- Centrifuge 10 mL of over-night liquid culture

- Resuspend pellet in 800 µL of resuspension solution

- Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)

- Centrifuge 3 min at 10,000 rpm

- Transfer 500 µL of supernatant into new 2 mL tube

- Add 500 µL of lysis buffer, invert 4 - 6 times

- Add 700 µL of neutralisation buffer, invert 4 - 6 times

- Centrifuge 10 min at 12,000 rpm

- Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)

- Two wash steps with 750 µL of washing buffer

- Dry column

- Elute in 75 µL of elution buffer

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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:







































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