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. Author manuscript; available in PMC: 2013 Feb 14.
Published in final edited form as: Nature. 2011 Jun 15;474(7351):337–342. doi: 10.1038/nature10163

Figure 3. Construction of Gateway-adapted intermediate targeting vectors by 96-well BAC recombineering.

Figure 3

Recombineering steps and elapsed time are shown. a, BAC clones, arrayed in 96-well format and electroporated with a plasmid expressing arabinose-inducible Red proteins (pBADgbaA)47.

b–d, After arabinose induction, cells are electroporated with PCR fragments containing R1-pheS/zeo-R2 Gateway element (b), loxP-kan-loxP cassette

(c) and R3-ori/ampR-R4 subcloning plasmid (d). e, After gap repair, plasmid DNA is prepared and transformed into Cre-expressing bacteria to remove the kanR cassette, leaving a single loxP site downstream of the critical exon. Antibiotics used at each step are: A, ampicillin; C, chloramphenicol; K, kanamycin; T, tetracycline; Z, zeocin.