Haynes:NewProtocol: Difference between revisions
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
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PCR mix<br> | PCR mix<br> | ||
15ul of Gibson Assembly Mix | 15ul of Gibson Assembly Mix | ||
(I will add the recipe soon, but for now, find 15ul aliquotes in PCR tubes in a clearly labeled bag in the -20C freezer door) | (I will add the recipe soon, but for now, find 15ul aliquotes in PCR tubes in a clearly labeled bag in the -20C freezer door... soonish) | ||
==Procedure== | ==Procedure== | ||
Latest revision as of 13:04, 26 October 2012
Gibson Assembly
Overview
The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps.
In the first step, 20 basepair overlaps are added to the sequences to be assembled:
In the second step, the PCR products are added to the Gibson Assembly Mix:
Materials
Designing primers:
PCR mix
15ul of Gibson Assembly Mix
(I will add the recipe soon, but for now, find 15ul aliquotes in PCR tubes in a clearly labeled bag in the -20C freezer door... soonish)
Procedure
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform thermocycling program
For now: http://django.gibthon.org/help/gibson/
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
- Gibson, DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) - Nature Methods 6(5) 343-5 PMID 19363495
Contact
- René at rene.davis at asu dot edu
or instead, discuss this protocol.
