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Team:Brown-Stanford/Lab/Protocols/Gibson
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== '''Gibson Assembly''' == | == '''Gibson Assembly''' == | ||
Revision as of 01:41, 10 July 2011
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Protocol Code: A1
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
