User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions
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'''Reaction set-up''' | '''Reaction set-up''' | ||
* Use 0.2 - 0.5 pmol, per NEB's suggestion | * Use 0.2 - 0.5 pmol, per NEB's suggestion, [http://www.neb.com/nebecomm/products/protocol819.asp] | ||
* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 | * pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 | ||
* Total volume of DNA = 5.0 μL max | * Total volume of DNA = 5.0 μL max | ||
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* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL | * Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL | ||
* Thermal cycler: 50°C,60 min.; 4°C, ∞ | * Thermal cycler: 50°C,60 min.; 4°C, ∞ | ||
* Transform | * Transform: add 20 μL to 50 μL DH5α-Turbo; ice/5 min.; plate on 100 μg/mL amp. | ||
Revision as of 17:35, 5 January 2013
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01/05/13
Gibson Assembly
Measure DNA Concentrations
1. hPCD + BL01 + V0120
2. hPCD + BL01 + V0120
Golden Gate Assembly
PCR
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