Team:Macquarie Australia/Protocols/GibsonAssembly

From 2013.igem.org

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Before beginning Gibson assembly, all agar plate requirements, concentration calculations and equipment requirements were prepared. Once the gBlocks had arrived Gibson assembly was performed on all fragments.<br><br>
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Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on <b>NEB guidelines</b>. </center>
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<b>NOTE:</b> For efficiency with reference to the NEB Gibson protocol, we considered:
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Each Gene Block fragment was supplied as 200ng and before use it was dissolved in 20µl of TE buffer. The table below shows each tube with vector & volume, volume of Gibson Master Mix, water volume & volume of each gBlock fragment. After incubation, these were transformed into Top10 cells using the Transformation Protocol found here - 
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<font size=3><b>1)</b></font> 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
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</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
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<font size=3><b>2)</b></font>  0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.
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A calculation provided was used furthermore to determine the pmols required based on the length of fragments: <br>
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<b>pmols</b> = (weight in ng) x 1,000 / (base pairs x 650 daltons)
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'''5. After addition of all components (shown in table) incubation was preformed at 50°C for 60 min followed. '''
 
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<td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
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.datagrid {border-collapse: collapse; text-align: left; width: 100%;} .datagrid {font: normal 12px/150% Verdana, Arial, Helvetica, sans-serif; background: #fff; overflow: hidden; }.datagrid table td, .datagrid table th { padding: 3px 12px; }.datagrid table thead th {background:-webkit-gradient( linear, left top, left bottom, color-stop(0.05, #991821), color-stop(1, #80141C) );background:-moz-linear-gradient( center top, #991821 5%, #80141C 100% );filter:progid:DXImageTransform.Microsoft.gradient(startColorstr='#991821', endColorstr='#80141C');background-color:#991821; color:#FFFFFF; font-size: 14px; font-weight: bold; border-left: 1px solid #B01C26; } .datagrid table thead th:first-child { border: none; }.datagrid table tbody td { color: #000000; border-left: 1px solid #DBDBDB;font-size: 12px;font-weight: normal; }.datagrid table tbody .alt td { background: #303030; color: #FFFFFF; }.datagrid table tbody td:first-child { border-left: none; }.datagrid table tbody tr:last-child td { border-bottom: none; }
 
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<center><font size=3><b>Legend</b></font></center>
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<table align="center"> <!-- Added by me -->
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<font size=5>*</font> =       50ng of 5000 bp dsDNA is approx 0.015 pmols.
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<thead><tr><th>Element </th><th>ChlM</th><th>ChlI1</th><th>CTH1</th><th>POR</th><th>ChlG</th><th>DVR1</th><th>ChlP</th><th>ChlI2</th><th>Gene</th><th>Gene</th></tr></thead>
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<tbody><tr><td>Fragment 1</td><td>3.3uL Gblock1</td><td>5.0uL Gblock1</td><td>2.3uL Gblock1</td><td>4.8uL Gblock1</td></tr>
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<font size=5>**</font> =  Control reagents
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<tr class="alt"><td>Fragment 2</td><td>3.3uL Gblock2</td><td>3.3uL Gblock2</td><td>3.2uL Gblock2</td><td>3.0uL Gblock2</td></tr>
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<tbody><tr><td>Fragment 3</td><td></td><td></td><td>3.1uL Gblock3</td><td></td></tr>
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<font size=5>***</font> =  Additional master mix may be required for larger bp fragments.  
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<tr class="alt"><td>10X T4 DNA ligase buffer</td><td>2 µL</td></tr>
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<tbody><tr><td>Vector (0.05pmol)</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td><td>2.7uL</td></tr>
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<b> Note - </b> 50ng of 500 bp is approx 0.15 pmol
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<tr class="alt"><td>H2O</td><td>0.7uL</td></tr>
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<tbody><tr><td>Gibson Master Mix</td><td>10 uL</td><td>11 uL</td><td>11.3 uL</td><td>10.5 uL</td></tr>
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<b> Note - </b>   50-100ng of vector recommended with excess insert of 2-3 fold
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<b> Note - </b>   Use 5 x more insert if  <200bps.
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<b>After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.</b>

Latest revision as of 10:15, 27 September 2013


Gibson Assembly Protocol


Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidelines.


NOTE: For efficiency with reference to the NEB Gibson protocol, we considered:

1) 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
2) 0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.

A calculation provided was used furthermore to determine the pmols required based on the length of fragments:
pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)

Legend

* = 50ng of 5000 bp dsDNA is approx 0.015 pmols.

** = Control reagents

*** = Additional master mix may be required for larger bp fragments.

Note - 50ng of 500 bp is approx 0.15 pmol

Note - 50-100ng of vector recommended with excess insert of 2-3 fold

Note - Use 5 x more insert if <200bps.


After addition of all components (shown in table) incubation was preformed in a thermocycler at 50°C for 60 min.

Retrieved from "http://2013.igem.org/Team:Macquarie_Australia/Protocols/GibsonAssembly"