Team:UNITN-Trento/Protocols

From 2013.igem.org

(Difference between revisions)
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{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|B. subtilis transformation (from Groeningen)|<html>
 +
<!--biobrick cloning protocol -->
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 +
<h1>BioBrick cloning</h1>
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<br/>
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<br/>
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<p>
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Prepare the digestion mix as follow:
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</p>
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<table>
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<tr>
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<td>
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DNA
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</td>
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<td>
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500 ng
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</td>
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</tr>
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<tr>
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<td>
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10X NEB Buffer
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</td>
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<td>
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2.5 ul
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</td>
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</tr>
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<tr>
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<td>
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10X BSA
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</td>
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<td>
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2.5 ul
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</td>
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</tr>
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<tr>
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<td>
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E1
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</td>
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<td>
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1 ul
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</td>
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</tr>
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<tr>
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<td>
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E2
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</td>
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<td>
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1 ul
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</td>
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</tr>
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<tr>
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<td>
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H2O
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</td>
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<td>
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Up to 25 ul
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</td>
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</tr>
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</table>
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 +
<p>
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Incubate the reaction mix at 37 &deg;C for 30 min. Disactivate then the enzymes incubating the mix at 80 &deg;C for 20 min.
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The next step will be the ligation of the digestion products. The raction mix is prepared as follow:
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</p>
 +
<table>
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<tr>
 +
<td>
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Insert
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</td>
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<td>
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3 fold excess
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</td>
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</tr>
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<tr>
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<td>
 +
Vector
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</td>
 +
<td>
 +
40 ng
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</td>
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</tr>
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<tr>
 +
<td>
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10X T4 Ligase Buffer
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</td>
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<td>
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2 ul
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</td>
 +
</tr>
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<tr>
 +
<td>
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T4 Ligase
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</td>
 +
<td>
 +
1 ul
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</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
H2O
 +
</td>
 +
<TD>
 +
Up to 20 ul
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</TD>
 +
</tr>
 +
 +
</table>
 +
<p>
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Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
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</p>
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</html>|subtilis-transformation}}
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 +
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Competent cells transformation efficiency kit (registry)|<html>
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<a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit" target="_blank">External link (registry)</a>
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</html>|Competent-cells-transformation-efficiency}}
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 +
<h2>Miscellaneous</h2>
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 +
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Parts extraction and transformation (NEB10&beta;).|<html>
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<ol>
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<li>Label the empty eppendorfs that will contain the parts, including antibiotic resistance, part denomination and position (and on which kit).</li>
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<li>Spot the correct well and label it with a pen.</li>
 +
<li>Push a hole with the pin of a micropipette and resuspend the content with 10ul water.</li>
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<li>When the color is dark red, wait 1 minute.</li>
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<li>Move the re-suspended part into the correct empty eppendorf.</li>
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</ol>
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</html>|parts-registry-extraction}}
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 +
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Wizard&reg; Plus SV Minipreps DNA Purification System Technical Bulletin|<html>
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<a href="http://ita.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Protocol.pdf" target="_blank">Complete protocol</a> (180kb)<br/>
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<a href="http://ita.promega.com/~/media/Files/Resources/ProtCards/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Quick%20Protocol.pdf" target="_blank">Quik protocol</a> (72kb)
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</html>|miniprep}}
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 +
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Biobrick cloning|<html>
<!--biobrick cloning protocol -->
<!--biobrick cloning protocol -->
<p>
<p>
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Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
Gently mix the reaction and incubate for 30 min at room temperature. Disactivate the enzymes at 80 &deg;C for 20 min. Transorm 10 ul of the reaction in competent cells.
</p>
</p>
-
</html>|subtilis-transformation}}
 
-
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Competent cells transformation efficiency kit (registry)|<html>
+
</html>|biobrick-cloning}}
-
<a href="http://parts.igem.org/Help:Transformation_Efficiency_Kit" target="_blank">External link (registry)</a>
+
-
</html>|Competent-cells-transformation-efficiency}}
+
-
 
+
-
<h2>Miscellaneous</h2>
+
-
 
+
-
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Parts extraction and transformation (NEB10&beta;).|<html>
+
-
<ol>
+
-
<li>Label the empty eppendorfs that will contain the parts, including antibiotic resistance, part denomination and position (and on which kit).</li>
+
-
<li>Spot the correct well and label it with a pen.</li>
+
-
<li>Push a hole with the pin of a micropipette and resuspend the content with 10ul water.</li>
+
-
<li>When the color is dark red, wait 1 minute.</li>
+
-
<li>Move the re-suspended part into the correct empty eppendorf.</li>
+
-
</ol>
+
-
</html>|parts-registry-extraction}}
+
-
 
+
-
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Wizard&reg; Plus SV Minipreps DNA Purification System Technical Bulletin|<html>
+
-
<a href="http://ita.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/0/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Protocol.pdf" target="_blank">Complete protocol</a> (180kb)<br/>
+
-
<a href="http://ita.promega.com/~/media/Files/Resources/ProtCards/Wizard%20Plus%20SV%20Minipreps%20DNA%20Purification%20System%20Quick%20Protocol.pdf" target="_blank">Quik protocol</a> (72kb)
+
-
</html>|miniprep}}
+
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Ligation|<html>
{{:Team:UNITN-Trento/Templates/Styles/ProtocolSpoiler|Ligation|<html>

Revision as of 12:32, 31 July 2013