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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.
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==Materials==
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For a 50 μL PCR reaction:
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* 35 μL H<sub>2</sub>O
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* 5 μL 10X PCR buffer
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* 5 μL 2mM dNTPs (each)
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* 1.5 μL 50mM MgCl<sub>2</sub>
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* 1 μL 50μM sense primer
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* 1 μL 50μM antisense primer
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* 1 μL 5nM DNA template
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* 0.5 μL TAQ DNA polyermerase
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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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## 95 °C 5 min
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## 95 °C 30 s
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## T<sub>H</sub> 30 s
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## 72 °C 1 min for each 1 kb PCR product
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## Repeat steps 2-4 a total of 12-36 times (24 is standard).
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## 72 °C 5 min
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## 12 °C hold
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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|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
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#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
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#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164
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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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-->

Revision as of 12:38, 26 October 2012

<- 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.

Materials

For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL TAQ DNA polyermerase

Procedure

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program
    1. 95 °C 5 min
    2. 95 °C 30 s
    3. TH 30 s
    4. 72 °C 1 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min
    7. 12 °C hold

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. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

Contact

  • René at rene.davis at asu dot edu

or instead, discuss this protocol.


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