Team:Tuebingen/Notebook/Protocols/3A assembly

From 2014.igem.org

(Difference between revisions)
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   <tr>
   <tr>
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     <td style="text-align: center">242 g</td>
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     <td style="text-align: center">500 ng</td>
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     <td>Tris</td>
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     <td>part plasmid DNA</td>
   </tr>
   </tr>
   <tr>
   <tr>
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     <td style="text-align: center">57.1 mL</td>
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     <td style="text-align: center">0.5 µL</td>
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     <td>pure acetic acid</td>
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     <td>of each restriction enzyme</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td style="text-align: center">0.5 M</td>
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     <td style="text-align: center">2.5 µL</td>
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     <td>EDTA 0.5 M pH 8.0</td>
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     <td>10x NEB buffer 2</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td style="text-align: center">0.25 µL</td>
 +
    <td>100x BSA</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td style="text-align: center">to 25 µL</td>
 +
    <td>H20</td>
   </tr>
   </tr>
</table>
</table>
<h4>Procedure</h4>
<h4>Procedure</h4>
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<p>Mix all the reagents and add to 1000 ml with distillated water. Adjust to pH 8.3. Store at room temperature.</p>
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<ol>
 +
  <li>After mixing the reagents in a 1,5 ml Eppendorf-tube: Incubation at 37 °C overnight.</li>
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  <li>Heat inactivate the restriction enzymes at 80 °C for 20 min.</li>
 +
</ol>
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<p>&nbsp;</p>
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 +
<h4><u>Ligation</u></h4>
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 +
<h4>Reagents</h4>
 +
<table border="0">
 +
  <colgroup>
 +
    <col width="100">
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    <col width="300">
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  </colgroup>
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 +
  <tr>
 +
    <td style="text-align: center">1 µL</td>
 +
    <td>10x ligation buffer</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">100-200 ng</td>
 +
    <td>upstream part plasmid</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">100-200 ng</td>
 +
    <td>downstream part plasmid</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">50 ng</td>
 +
    <td>destination part plasmid</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">1 µL</td>
 +
    <td>T4 ligase</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">1 µL</td>
 +
    <td>ATP</td>
 +
  </tr>
 +
 +
  <tr>
 +
    <td style="text-align: center">to 10 µL</td>
 +
    <td>H20</td>
 +
  </tr>
 +
</table>
 +
 +
<h4>Procedure</h4>
 +
<ol>
 +
  <li>Mix all reagents in a PCR-tube and if necessary add to 10 μl with H20.</li>
 +
  <li>Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.</li>
 +
  <li>Heat inactivate the enzymes for 5 min at 70 °C.</li>
 +
  <li>Store at -20 °C.</li>
 +
</ol>

Revision as of 23:25, 14 October 2014


Three part assembly

Digestion

Reagents

500 ng part plasmid DNA
0.5 µL of each restriction enzyme
2.5 µL 10x NEB buffer 2
0.25 µL 100x BSA
to 25 µL H20

Procedure

  1. After mixing the reagents in a 1,5 ml Eppendorf-tube: Incubation at 37 °C overnight.
  2. Heat inactivate the restriction enzymes at 80 °C for 20 min.

 

Ligation

Reagents

1 µL 10x ligation buffer
100-200 ng upstream part plasmid
100-200 ng downstream part plasmid
50 ng destination part plasmid
1 µL T4 ligase
1 µL ATP
to 10 µL H20

Procedure

  1. Mix all reagents in a PCR-tube and if necessary add to 10 μl with H20.
  2. Run cycler starting at 25 °C, reducing temperature hourly by 1 degree and remaining at 4 °C.
  3. Heat inactivate the enzymes for 5 min at 70 °C.
  4. Store at -20 °C.

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