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Team:Cambridge/Gibson/Protocol
From 2010.igem.org
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Master Mix is 1.33x concentrated | Master Mix is 1.33x concentrated | ||
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*Add DNA to be ligated and master mix in volumetric ratio 1:3 | *Add DNA to be ligated and master mix in volumetric ratio 1:3 | ||
| + | *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> | <i>e.g. If you were ligating two fragments (A and B) you could put:</i> | ||
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Revision as of 13:16, 30 September 2010
Gibson Assembly: Protocols
The formal paper in nature describing Gibson Assembly can be found 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 at the point of ligation.
- The standard way to do this is with PCR with specialised primers
- We have designed a tool to help you do this: 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
- Prepare Master Mix
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 |
Master Mix is 1.33x concentrated
- Add DNA to be ligated and master mix in volumetric ratio 1:3
- Incubate for 1 hour at 50°C
e.g. If you were ligating two fragments (A and B) you could put:
| 2.5µl | fragment A |
| 2.5µl | fragment B |
| 15µl | Gibson Master Mix |
