Lidstrom:Gibson Assembly
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Janet's Tips
Janet is developing a protocol here
Design:
Fragments
- overlap by 40 bp
- efficiency
Oligo (primer) Design
PCR
Assembly
Transformation
- Electroporation can give much higher efficiency. For this reason, the assembled product is usually electroporated.
- rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells.
- Janet electroporates 2 uL
Screening
- Colony PCR 1st
- Sequence a few clones with primers that cover the "seams."
Misc:
- Electrocompetence is ~ 1000x more effective so is tempting. -
