Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination
Figure 6
(a) Strategy for library screening through homologous recombination. There are 3 steps: (I) Library construction—consists of DNA ligation and E. coli transformation; (II) Library amplification—library is plated on Cm agar plates at high density, clones harvested by washing with LB. Glycerol stocks made of mixed clones; (III) Library screening via HR. An aliquot of the amplified library is grown at 32°C and induced at 42°C. Electrocompetent cells are prepared and transformed with HR cassette (Figure 6(b)). Recombinant clones were identified on kanamycin plates. (b) Recombination cassette. HR cassette consists of a 1.3 Kb kanamycin resistance gene flanked by 50 bp homology arms. cassette is inserted into targeting gene using 50 bp homology arms “H” corresponding to the targeted region. “F” and “Rev” are the flanking screening primers used for screening recombinant clones.
