Haynes:NewProtocol
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| - | + | <- [[Haynes:Protocols | Back to Protocols]] | |
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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 13:38, 26 October 2012
Gibson Assembly
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
- 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.
