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- overlap by 40 bp
Oligo (primer) Design
- 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 hasn't had a problem transforming 1 uL of product if a cuvette is new. Older cuvettes arc more easily.
- Colony PCR 1st
- Sequence a few clones with primers that cover the "seams."
- Electrocompetence is ~ 1000x more effective so is tempting. -