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Team:Macquarie Australia/Protocols/GibsonAssembly
From 2013.igem.org
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| - | NOTE: For efficiency with reference to the NEB Gibson protocol, we considered: | + | <b>NOTE:</b> For efficiency with reference to the NEB Gibson protocol, we considered: |
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| - | < | + | <table border="0" cellpadding="10"> |
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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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| + | <center><font size=3><b>Legend</b></font></center> | ||
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| + | <font size=5>*</font> = 50ng of 5000 bp dsDNA is approx 0.015 pmols. | ||
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| - | <font size= | + | <font size=5>**</font> = Control reagents |
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| - | <font size= | + | <font size=5>***</font> = Additional master mix may be required for larger bp fragments. |
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| - | + | <b> Note - </b> 50ng of 500 bp is approx 0.15 pmol | |
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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> | <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
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)
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* = 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.
