Haynes:Gibson Assembly: Difference between revisions
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<font size=3>'''Gibson Assembly'''</font><br> | <font size=3>'''Gibson Assembly'''</font><br> | ||
by René Davis, 2012</div> | |||
<br> | <br> | ||
==Overview== | ==Overview== | ||
<div style="width: 800px"> | |||
The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps.<br> | |||
In the first step, 20 basepair overlaps are added to the sequences to be assembled. In the example below, I am assembling two proteins joined by a 60 basepair linker: | |||
[[image:Gibson assembly image 1.png|500px|Gibson Assembly step 1]] <br> | |||
In the second step, the PCR products are added to the Gibson Assembly Mix: | |||
[[image:Slide2.jpg|500px|Gibson Assembly step 2]] | |||
==Materials== | ==Materials== | ||
Designing primers: | |||
[[Image:Slide_3_pdf.png|500px|Gibson Assembly primers]]<br> | |||
PCR mix<br> | |||
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== | ==Procedure== | ||
# In a PCR tube, mix the components on ice in the order they are listed above. | # In a PCR tube, mix the components on ice in the order they are listed above. | ||
# Perform thermocycling program | # Perform thermocycling program | ||
## | ## | ||
For now: http://django.gibthon.org/help/gibson/ | |||
==Notes== | ==Notes== | ||
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</biblio>--> | </biblio>--> | ||
<!-- Try the [[Template:FormatRef|FormatRef template]]--> | <!-- Try the [[Template:FormatRef|FormatRef template]]--> | ||
#{{FormatRef| | #{{FormatRef|Gibson, DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO |2009| |Nature Methods 6(5)|343-5| }} PMID 19363495 | ||
==Contact== | ==Contact== | ||
* | *René at rene.davis at asu dot edu | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. |
Latest revision as of 12:06, 25 February 2014
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 example below, I am assembling two proteins joined by a 60 basepair linker:
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.