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<div style="width: 800px" align="center"> | |||
<font size=3>'''Gibson Assembly'''</font><br> | |||
By René Davis, 2012</div> | |||
<br> | |||
==Overview== | |||
<div style="width: 800px"> | |||
The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps. | |||
==Materials== | |||
For a 50 μL PCR reaction: | |||
* 35 μL H<sub>2</sub>O | |||
* 5 μL 10X PCR buffer | |||
* 5 μL 2mM dNTPs (each) | |||
* 1.5 μL 50mM MgCl<sub>2</sub> | |||
* 1 μL 50μM sense primer | |||
* 1 μL 50μM antisense primer | |||
* 1 μL 5nM DNA template | |||
* 0.5 μL TAQ DNA polyermerase | |||
==Procedure== | |||
# In a PCR tube, mix the components on ice in the order they are listed above. | |||
# Perform thermocycling program | |||
## 95 °C 5 min | |||
## 95 °C 30 s | |||
## T<sub>H</sub> 30 s | |||
## 72 °C 1 min for each 1 kb PCR product | |||
## Repeat steps 2-4 a total of 12-36 times (24 is standard). | |||
## 72 °C 5 min | |||
## 12 °C hold | |||
==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 <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip. | |||
==References== | |||
'''Relevant papers and books''' | |||
<!-- If this protocol has papers or books associated with it, list those references here.--> | |||
<!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently. | |||
<biblio> | |||
#Goldbeter-PNAS-1981 pmid=6947258 | |||
#Jacob-JMB-1961 pmid=13718526 | |||
#Ptashne-Genetic-Switch isbn=0879697164 | |||
</biblio>--> | |||
<!-- Try the [[Template:FormatRef|FormatRef template]]--> | |||
#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258 | |||
#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526 | |||
#{{FormatRef|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, [[Talk:{{PAGENAME}}|discuss this protocol]]. | |||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | |||
<!-- Move the relevant categories above this line to tag your protocol with the label | |||
[[Category:Protocol]] | |||
[[Category:Needs attention]] | |||
[[Category:In vitro]] | |||
[[Category:In vivo]] | |||
[[Category:In silico]] | |||
[[Category:DNA]] | |||
[[Category:RNA]] | |||
[[Category:Protein]] | |||
[[Category:Chemical]] | |||
[[Category:Escherichia coli]] | |||
[[Category:Yeast]] | |||
--> | |||
Revision as of 10:38, 26 October 2012
Gibson Assembly
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
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform thermocycling program
- 95 °C 5 min
- 95 °C 30 s
- TH 30 s
- 72 °C 1 min for each 1 kb PCR product
- Repeat steps 2-4 a total of 12-36 times (24 is standard).
- 72 °C 5 min
- 12 °C hold
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
- Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
- Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
- 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.
