Team:Macquarie Australia/Protocols/GibsonAssembly

From 2013.igem.org

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Gibson assembly was employed to combine homologous ends of various gBlocks and PCR fragments based on NEB guidlines.  
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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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<font size=5>•</font> 0.02–0.2 pmols of DNA fragments when 2 or 3 different fragments were being assembled.
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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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<font size=5>•</font> 0.2–1 pmols of DNA when 4 to 6 different fragments were being assembled.  
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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:
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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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pmols = (weight in ng) x 1,000 / (base pairs x 650 daltons)
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<b>pmols</b> = (weight in ng) x 1,000 / (base pairs x 650 daltons)
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The cells were appropriately transformed and concentrations were determined prior. (transformation protocol can be found with the following link)
 
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-</html>[[Team:Macquarie/Protocols/TransformationProtocol|Transformation Protocol]]. <html></p></html>
 
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<center><h1><td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
<center><h1><td><img src="https://static.igem.org/mediawiki/2013/d/d6/Gibson_protocol.jpg" width=550 height=336></td>
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*      50ng of 5000 bp dsDNA is approx 0.015 pmols.
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<center><font size=3><b>Legend</b></font>
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<p></p> - 50ng of 500 bp is approx 0.15 pmol
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<font size=3><b>*</b></font>      50ng of 5000 bp dsDNA is approx 0.015 pmols.
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<p></p> - 50-100ng of vector recommended with excess insert of 2-3 fold
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<font size=3><b>**</b></font>    Control reagents
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<p></p> - Use 5 x more insert if  <200bps.
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<font size=3><b>***</b></font>  Additional master mix may be required for larger bp fragments.  
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**  Control reagents
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<font size=3><b>-</b></font>  50ng of 500 bp is approx 0.15 pmol
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*** Additional master mix may be required for larger bp fragments.
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<font size=3><b>-</b></font>  50-100ng of vector recommended with excess insert of 2-3 fold
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<font size=3><b>-</b></font>  Use 5 x more insert if  <200bps.
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After addition of all components (shown in table) incubation was preformed in thermocycler at 50°C for 60 min.
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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>

Revision as of 10:05, 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.

- 50ng of 500 bp is approx 0.15 pmol

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

- 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"