Team:Tuebingen/Notebook/Protocols/3a-assembly

From 2013.igem.org

(Difference between revisions)
Line 35: Line 35:
   <tr>
   <tr>
-
     <td style="text-align: center">0.25 µL</td>
+
     <td style="text-align: center">0.25</td>
     <td>100x BSA</td>
     <td>100x BSA</td>
   </tr>
   </tr>
Line 68: Line 68:
<h3>Procedure</h3>
<h3>Procedure</h3>
-
<p>Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night. On the next day, heat inactivate restriction enzymes at 80°C for 20 min. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions. Heat inactivate phosphatase at 70 °C for 5 min.</p>
+
<ol>
-
 
+
  <li>Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.</li>
-
<p>Run <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/gelelectrophoresis">gel</a> and continue with <a href="https://2013.igem.org/Team:Tuebingen/Notebook/Protocols/gelextraction">gelextraction</a>.</p>
+
  <li>On the next day, heat inactivate restriction enzymes at 80°C for 20 min.</li>
 +
  <li>Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions.</li>
 +
  <li>Heat inactivate phosphatase at 70 °C for 5 min.</li>
 +
  <li>Perform the previous steps for upstream part plasmid, downstream part plasmid, and destination part plasmid. Digest upstream part with EcoRI / SpeI, downstream part with XbaI / PstI, and destination part plasmid with EcoRI / PstI.</li>
 +
<p>&nbsp;</p>
-
<h2>Ligation</h2>
+
<h2>Digestion</h2>
</div>
</div>

Revision as of 01:47, 4 October 2013

Return to iGEM Main Page.

3A-Assembly
Back to Protocols

 

Digestion

Reagents

2.5 µL 10x NEBuffer 2
0.25 100x BSA
250 - 300 ng Plasmid DNA
0.5 µL EcoRI or XbaI
0.5 µL PstI or SpeI
to 25 µL Aqua dest.
1/10 vol 10X Antarctic Phosphatase

 

Procedure

  1. Mix all reagents (except for Antarctic Phosphatase) in an 1.5 mL Eppendorf-tube and incubate at 37 °C over night.
  2. On the next day, heat inactivate restriction enzymes at 80°C for 20 min.
  3. Add 1/10 volume of 10X Antarctic Phosphatase and mix. Incubate at 37 °C for 15 min for 5' extensions.
  4. Heat inactivate phosphatase at 70 °C for 5 min.
  5. Perform the previous steps for upstream part plasmid, downstream part plasmid, and destination part plasmid. Digest upstream part with EcoRI / SpeI, downstream part with XbaI / PstI, and destination part plasmid with EcoRI / PstI.

    6.  

    Digestion


Retrieved from "http://2013.igem.org/Team:Tuebingen/Notebook/Protocols/3a-assembly"