Team:Cambridge/Gibson/Protocol

From 2010.igem.org

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{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: Protocol}}
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The formal paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].
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The original paper in nature describing Gibson Assembly can be found [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html here].
==Step 1: Design Primers==
==Step 1: Design Primers==
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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School}}
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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School - Try out our new and improved [[Team:Cambridge/Tools/Gibson | Gibthon]] Oligo design}}
<div style="float:right; clear:both">&nbsp;</div>
<div style="float:right; clear:both">&nbsp;</div>
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{{:Team:Cambridge/Templates/RightImage|image=Gibthon.png|caption=New and improved Gibthon oligo design}}
 
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
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The standard way to do this is with PCR with specialised primers. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
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The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
==Step 2: Order Primers==
==Step 2: Order Primers==
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==Step 3: PCR ==
==Step 3: PCR ==
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PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer:
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<div style="float:right; clear:both">&nbsp;</div>
<div style="float:right; clear:both">&nbsp;</div>
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}
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PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.
 
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{|class="wikitable"
 
-
|-
 
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|Step
 
-
|Temp
 
-
|Time
 
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|-
 
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|1:Initial Melting
 
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|98°C
 
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|30s
 
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|-
 
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|2:Melting
 
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|98°C
 
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|10s
 
-
|-
 
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|3:Annealing
 
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|T<sub>m</sub>°C
 
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|15s
 
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|-
 
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|4:Elongation
 
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|72
 
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|45s per kb DNA
 
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|-
 
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|5:GoTo step 2
 
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|
 
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|30 times
 
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|-
 
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|6:Final Elongation
 
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|72°C
 
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|7m30
 
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|-
 
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|7:Final Hold
 
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|4°C
 
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|∞
 
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|}
 
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-
 
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<html>
<html>
<style>
<style>
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
</style>
</style>
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<div align="center">
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<div align="left">
<table class="vistable" padding="0" cellspacing="0">  
<table class="vistable" padding="0" cellspacing="0">  
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 +
<tr>
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<td>Step
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</td><td>Temp
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</td><td>Time
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</td></tr>
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<tr>
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<td>1:Initial Melting
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</td><td>98°C
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</td><td>30s
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</td></tr>
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<tr>
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<td>2:Melting
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</td><td>98°C
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</td><td>10s
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</td></tr>
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<tr>
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<td>3:Annealing
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</td><td>T<sub>m</sub>°C
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</td><td>15s
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</td></tr>
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<tr>
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<td>4:Elongation
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</td><td>72
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</td><td>45s per kb DNA
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</td></tr>
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<tr>
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<td>5:GoTo step 2
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</td><td>
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</td><td>30 times
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</td></tr>
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<tr>
 +
 +
<td>6:Final Elongation
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</td><td>72°C
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</td><td>7m30
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</td></tr>
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<tr>
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<td>7:Final Hold
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</td><td>4°C
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</td><td>∞
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</td></tr>
   
   
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<tr>
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<td><b>Assembly Method</b>
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</td><td><b>Number of steps to ligate N pieces of DNA</b>
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</td></tr>
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</table>  
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<tr>
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<td><a href="http://partsregistry.org/Assembly:Standard_assembly" class="external text" title="http://partsregistry.org/Assembly:Standard_assembly" rel="nofollow">Standard Assembly</a>
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</td><td><a href="/Image:Steps-N.png" class="image" title="Image:Steps-N.png"><img alt="Image:Steps-N.png" src="/wiki/images/c/cc/Steps-N.png"  border="0" /></a>
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-
</td></tr>
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<tr>
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<td><a href="http://partsregistry.org/Assembly:Rolling_assembly" class="external text" title="http://partsregistry.org/Assembly:Rolling_assembly" rel="nofollow">Parallel Assembly</a>
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</td><td><a href="/Image:Steps-Log2.png" class="image" title="Image:Steps-Log2.png"><img alt="Image:Steps-Log2.png" src="/wiki/images/6/6f/Steps-Log2.png" width="138" height="21" border="0" /></a> (rounded up)
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</td></tr>
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<tr>
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<td><a href="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" class="external text" title="https://2010.igem.org/Team:Cambridge/Gibson/Mechanism" rel="nofollow">Gibson Assembly</a>
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</td><td><a href="/Image:Steps1.png" class="image" title="Image:Steps1.png"><img alt="Image:Steps1.png" src="/wiki/images/1/10/Steps1.png" width="83" height="18" border="0" /></a>
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-
</td></tr></table>  
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</div>
</div>
</html>
</html>
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The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
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<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
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{|class="wikitable"
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<html>
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|-
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<style>
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|<i>2.5µl</i>
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table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}
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|<i>fragment A</i>
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
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|-
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</style>
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|<i>2.5µl</i>
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<div align="left">
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|<i>fragment B</i>
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<table class="vistable" padding="0" cellspacing="0">
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|-
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<tr>
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|<i>15µl</i>
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<td><i>2.5µl</i>
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|<i>Gibson Master Mix</i>
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</td><td><i>fragment A</i>
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|}
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</td></tr>
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<tr>
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<td><i>2.5µl</i>
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</td><td><i>fragment B</i>
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</td></tr>
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<tr>
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<td><i>15µl</i>
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</td><td><i>Gibson Master Mix</i>
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</td></tr>
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</table>
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</div>
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</html>
==Step 5: Transformation==
==Step 5: Transformation==

Latest revision as of 20:31, 26 October 2010

Retrieved from "http://2010.igem.org/Team:Cambridge/Gibson/Protocol"