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Team:Brown-Stanford/Lab/Protocols/Gibson
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(Created page with "{{:Team:Brown-Stanford/Templates/Main}} == '''Gibson Assembly''' == '''5x isothermal buffer''' *0.75g - 25% PEG-8000 *1500 µL - 500 mM Tris-HCl pH 7.5 *75 µL - 50mM M...") |
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*Nuclease-free water to fill to 3000µL | *Nuclease-free water to fill to 3000µL | ||
'''Total - 3000 µL''' | '''Total - 3000 µL''' | ||
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| + | '''Gibson Master Mix''' | ||
| + | *50 µL - Taq ligase (40u/µL) | ||
| + | *100 µL - 5x isothermal buffer | ||
| + | *2 µL - T5 exonuclease (1u/µL) | ||
| + | *6.25 µL - Phusion polymerase (2u/µL) | ||
| + | *216.75 µL - Nuclease-free water | ||
| + | '''Total - 375 µL''' | ||
{{:Team:Brown-Stanford/Templates/Foot}} | {{:Team:Brown-Stanford/Templates/Foot}} | ||
Revision as of 21:24, 30 June 2011
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Gibson Assembly
5x isothermal buffer
- 0.75g - 25% PEG-8000
- 1500 µL - 500 mM Tris-HCl pH 7.5
- 75 µL - 50mM MgCl2
- 150 µL - 50mM DTT
- 30 µL - 1mM dATP
- 30 µL - 1mM dTTP
- 30 µL - 1mM dCTP
- 30 µL - 1mM dGTP
- 300 µL - 5mM NAD
- Nuclease-free water to fill to 3000µL
Total - 3000 µL
Gibson Master Mix
- 50 µL - Taq ligase (40u/µL)
- 100 µL - 5x isothermal buffer
- 2 µL - T5 exonuclease (1u/µL)
- 6.25 µL - Phusion polymerase (2u/µL)
- 216.75 µL - Nuclease-free water
Total - 375 µL
