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Team:Brown-Stanford/Lab/Protocols/Gibson
From 2011.igem.org
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'''5x isothermal buffer''' | '''5x isothermal buffer''' | ||
*0.75g - 25% PEG-8000 | *0.75g - 25% PEG-8000 | ||
| - | *1500 µL - | + | *1500 µL - 1mM Tris-HCl pH 7.5 |
| - | *75 µL - | + | *75 µL - 1mM MgCl<sub>2</sub> |
| - | *150 µL - | + | *150 µL - 1mM DTT |
| - | *30 µL - | + | *30 µL - 100mM dATP |
| - | *30 µL - | + | *30 µL - 100mM dTTP |
| - | *30 µL - | + | *30 µL - 100mM dCTP |
| - | *30 µL - | + | *30 µL - 100mM dGTP |
| - | *300 µL - | + | *300 µL - 50mM NAD |
*Nuclease-free water to fill to 3000µL | *Nuclease-free water to fill to 3000µL | ||
'''Total - 3000 µL''' | '''Total - 3000 µL''' | ||
Revision as of 00:07, 29 July 2011
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REGObricks
Intro--------------------- ISRU--------------------- Biocement--------------------- S. pasteurii--------------------- Balloon
Flights--------------------- Transforming--------------------- Biobrick2 -
PowerCell
Intro--------------------- Cyanobacteria--------------------- Mars--------------------- Nutrients -
FRETcetera
-
Lab
-
Partners
-
Outreach
SB5.0--------------------- NASA--------------------- Lunar Science--------------------- Maker Faire--------------------- BBC--------------------- CBricks--------------------- Ethics--------------------- Regional
Jamboree
Protocol Code: AY1
Gibson Assembly
5x isothermal buffer
- 0.75g - 25% PEG-8000
- 1500 µL - 1mM Tris-HCl pH 7.5
- 75 µL - 1mM MgCl2
- 150 µL - 1mM DTT
- 30 µL - 100mM dATP
- 30 µL - 100mM dTTP
- 30 µL - 100mM dCTP
- 30 µL - 100mM dGTP
- 300 µL - 50mM 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
