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New Protocol
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<- [[Haynes:Protocols | Back to Protocols]]
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<div style="width: 800px" align="center">
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<font size=3>'''Gibson Assembly'''</font><br>
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By René Davis, 2012</div>
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<br>
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==Overview==
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<div style="width: 800px">
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The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps.<br>
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In the first step, 20 basepair overlaps are added to the sequences to be assembled:
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[[image:Slide1.png|500px|Gibson Assembly step 1]] <br>
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In the second step, the PCR products are added to the Gibson Assembly Mix:
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[[image:Slide2.jpg‎|500px|Gibson Assembly step 2]]
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==Materials==
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Designing primers:
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[[Image:Slide_3_pdf.png‎|500px|Gibson Assembly primers]]<br>
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PCR mix<br>
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15ul of Gibson Assembly Mix
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(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)
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==Procedure==
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# In a PCR tube, mix the components on ice in the order they are listed above.
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# Perform thermocycling program
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##
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For now: http://django.gibthon.org/help/gibson/
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==Notes==
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Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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#List troubleshooting tips here. 
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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#Anecdotal observations that might be of use to others can also be posted here. 
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==References==
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'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here.-->
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<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>-->
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<!-- Try the [[Template:FormatRef|FormatRef template]]-->
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#{{FormatRef|Gibson, DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO |2009| |Nature Methods 6(5)|343-5| }} PMID 19363495
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==Contact==
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*René at rene.davis at asu dot edu
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
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<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
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[[Category:Protocol]]
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[[Category:Needs attention]]
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[[Category:In vitro]]
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[[Category:In vivo]]
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[[Category:In silico]]
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[[Category:DNA]]
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[[Category:RNA]]
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[[Category:Protein]]
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[[Category:Chemical]]
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[[Category:Escherichia coli]]
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[[Category:Yeast]]
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-->

Current revision

<- Back to Protocols

Gibson Assembly

By René Davis, 2012


Contents

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:

Gibson Assembly step 1

In the second step, the PCR products are added to the Gibson Assembly Mix:

Gibson Assembly step 2

Materials

Designing primers:

Gibson Assembly 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

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. 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!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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

  1. 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.


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