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Team:Cambridge/Gibson/Protocol
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{{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Protocols}} | {{:Team:Cambridge/Templates/headerbar|colour=#96d446|title=Gibson Assembly: Protocols}} | ||
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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]. | ||
==Step 1) Design Primers== | ==Step 1) Design Primers== | ||
Revision as of 12:39, 30 September 2010
Gibson Assembly: Protocols
The formal 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
- If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap with respect to each other.
//Insert diagram of two pieces of DNA with overlap
- 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]
Step 2) Order Primers
- This step can take a while, so Gibson Assembly requires some planning ahead
Step 3) PCR
Step 4) Gibson Assembly
Master mix
| Volume/µl | |
| Taq ligase (40u/µl) | 50 |
| 5x isothermal buffer | 100 |
| T5 exonuclease (1u/µl) | 2 |
| Phusion polymerase (2u/µl) | 6.25 |
| Nuclease-free water | 216.75 |
| =375 |
