Team:Cambridge/Gibson/RFC

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{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: RFC}}
{{:Team:Cambridge/Templates/headerbar|colour=#fb5c2b|title=Gibson Assembly: RFC}}
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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-RFC.JPG|caption=[http://www.cambridgeigem.org/RFC57.pdf RFC 57], submitted by the Cambridge team}}
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The [http://bbf.openwetware.org/ BioBrick Foundation] uses documents known as [http://bbf.openwetware.org/RFC.html RFC]s to document standard techniques in synthetic biology.  Until now no such RFC existed for Gibson Assembly.  The Cambridge team created [http://www.cambridgeigem.org/RFC57.pdf '''RFC 57'''] to provide standard protocols for the technique.
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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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==Step 1: Design Primers==
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Gibson assembly allows joining any sequences using appropriate overlaps. As no restriction enzymes are used, this assembly method technically does not require the assembled parts to be in BioBrick format. It must however be emphasised that it is still necessary to comply with existing RFCs in terms of prefixes, suffixes and illegal sites for all new parts that are entered into the registry. Gibson assembly can be used as an alternative to BioBrick Standard Assembly, while retaining full backwards compatibility.  
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{{:Team:Cambridge/Templates/RightImage|image=Cambridge-oligoface.jpg|caption=Designing Oligos Old-School}}
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<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}}
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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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==Step 2: Order Primers==
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This step can take a while, so Gibson Assembly requires some planning ahead
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==Step 3: PCR ==
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<div style="float:right; clear:both">&nbsp;</div>
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{{: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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<html>
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<style>
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table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
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</style>
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<div align="center">
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<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>
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<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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</table>
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</div>
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</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.
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The Polymerase mixture we used was [http://www.finnzymes.com/pcr/phusion_high_fidelity_pcr_mastermix.html 2x Phusion MasterMix].
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==Step 4: Gibson Assembly==
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1) Prepare [[Team:Cambridge/Gibson/MasterMix | Gibson Master Mix]]
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2) Add DNA to be ligated and Master Mix in volumetric ratio 1:3
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3) Incubate for 1 hour at 50°C
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<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
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<html>
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<style>
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table.vistable td{border-right:1px solid gray; border-top:1px solid gray; padding:10px;}
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table.vistable{border-left:1px solid gray; border-bottom:1px solid gray;}
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</style>
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<div align="center">
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<table class="vistable" padding="0" cellspacing="0">
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<tr>
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<td><i>2.5µl</i>
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</td><td><i>fragment A</i>
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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>
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==Step 5: Transformation==
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The reaction mixture generated above should contain enough DNA to directly transform cells, although this is of course limited by the amount of DNA in the tube before the ligation.
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Latest revision as of 16:57, 27 October 2010

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