PCR was performed using either Phusion U Green Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Host Start High-Fidelity 2x Master Mix (New England Biolabs) unless otherwise noted. All plasmids were assembled by either the USER cloning method as previously described20 (link) or by Gibson assembly21 (link). Plasmids for mammalian cell transfections were prepared using an endotoxin removal plasmid purification system, ZymoPURE Plasmid Midiprep (Zymo Research Corporation).
Protocol detail
Efficient Plasmid Preparation and PCR
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Phusion U Green Multiplex PCR Master Mix
Q5 Host Start High-Fidelity 2x Master Mix
ZymoPURE Plasmid Midiprep
Cell
Endotoxin
Mammalian
Multiplex pcr
Plasmid
Transfections
Corresponding Organization :
Other organizations : Broad Institute, Howard Hughes Medical Institute, Harvard University
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Variable analysis
independent variables
- Choice of PCR master mix (Phusion U Green Multiplex PCR Master Mix or Q5 Host Start High-Fidelity 2x Master Mix)
- Not explicitly mentioned
- Plasmid assembly method (USER cloning or Gibson assembly)
- Plasmid purification method (ZymoPURE Plasmid Midiprep)
- Not specified
- Not specified
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