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Team:Cambridge/Gibson/Protocol
From 2010.igem.org
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==Step 1) Design Primers== | ==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 | + | *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 | *The standard way to do this is with PCR with specialised primers | ||
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==Step 4) Gibson Assembly== | ==Step 4) Gibson Assembly== | ||
| - | + | *Prepare Master Mix | |
| - | + | === Master mix === | |
| - | + | ||
| - | + | ||
| - | == Master mix == | + | |
{|class="wikitable" | {|class="wikitable" | ||
| Line 50: | Line 46: | ||
|=375 | |=375 | ||
|} | |} | ||
| + | Master Mix is 1.33x concentrated | ||
| + | |||
| + | *Add DNA to be ligated and master mix in volumetric ratio 1:3 | ||
| + | <i>e.g. If you were ligating two fragments (A and B) you could put | ||
| + | {|class="wikitable" | ||
| + | |- | ||
| + | |2.5µl | ||
| + | |fragment A | ||
| + | |- | ||
| + | |2.5µl | ||
| + | |fragment B | ||
| + | |- | ||
| + | |15µl | ||
| + | |Gibson Master Mix | ||
| + | |} | ||
| + | </i> | ||
{{:Team:Cambridge/Templates/footer}} | {{:Team:Cambridge/Templates/footer}} | ||
Revision as of 13:13, 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 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
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
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 |
