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Team:Cambridge/Gibson/Protocol
From 2010.igem.org
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|Time | |Time | ||
|- | |- | ||
| - | |1 | + | |1:Initial Melting |
|98°C | |98°C | ||
|30s | |30s | ||
|- | |- | ||
| - | | | + | |2:Melting |
| - | | | + | |98°C |
| - | | | + | |10s |
|- | |- | ||
| + | |3:Annealing | ||
| + | |T<sub>m</sub>°C | ||
| + | |15s | ||
| + | |- | ||
| + | |4:Elongation | ||
| + | |72 | ||
| + | |45s/kb | ||
| + | |- | ||
| + | |5:GoTo step 2 | ||
| | | | ||
| - | | | + | |30 times |
| - | | | + | |- |
| + | |6:Final Elongation | ||
| + | |72°C | ||
| + | |7m30 | ||
| + | |- | ||
| + | |7:Final Hold | ||
| + | |4°C | ||
| + | |∞ | ||
|} | |} | ||
Revision as of 20:14, 23 October 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 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: [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
- PCR is a dark art, but we have found that these general principles have served us well over the summer.
- 98°C 30s
| Step | Temp | Time |
| 1:Initial Melting | 98°C | 30s |
| 2:Melting | 98°C | 10s |
| 3:Annealing | Tm°C | 15s |
| 4:Elongation | 72 | 45s/kb |
| 5:GoTo step 2 | 30 times | |
| 6:Final Elongation | 72°C | 7m30 |
| 7:Final Hold | 4°C | ∞ |
Step 4: Gibson Assembly
- Prepare Master Mix
- 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 |
Step 5: Transformation
