Haynes:Gibson Assembly

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<font size=3>'''Gibson Assembly'''</font><br>
<font size=3>'''Gibson Assembly'''</font><br>
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By René Davis, 2012</div>
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by René Davis, 2012</div>
<br>
<br>
==Overview==
==Overview==
<div style="width: 800px">
<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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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. In the example below, I am assembling two proteins joined by a 60 basepair linker:
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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]]
==Materials==
==Materials==
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Designing primers:
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For a 50 μL PCR reaction:
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[[Image:Slide_3_pdf.png‎|500px|Gibson Assembly primers]]<br>
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* 35 μL H<sub>2</sub>O
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PCR mix<br>
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* 5 μL 10X PCR buffer
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15ul of Gibson Assembly Mix
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* 5 μL 2mM dNTPs (each)
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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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* 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==
==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
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## 95 °C 5 min
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##
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## 95 °C 30 s
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## T<sub>H</sub> 30 s
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For now: http://django.gibthon.org/help/gibson/
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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==
==Notes==
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</biblio>-->
</biblio>-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
<!-- 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|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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#{{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==
==Contact==
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*Who has experience with this protocol?
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*René at rene.davis at asu dot edu
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Revision as of 19:20, 29 October 2012

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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. In the example below, I am assembling two proteins joined by a 60 basepair linker:

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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