Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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One-Step BAC Modification: Preparation of Plasmids

(Protocol summary only for purposes of this preview site)

In the one-step approach, two plasmids are introduced into the BAC host cells (see Figs. 7 and 8 in the chapter introduction). The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the protein to replicate, must be grown in PIR1- or PIR2-competent E. coli. Our preference for these vectors is PIR1, because these cells are able to maintain about 250 copies of the donor vector. This small-sized vector is stable in PIR1. The RecA plasmid pSV1.RecA has a temperature-sensitive origin of replication and can be grown in most competent bacteria at 30C; here we use DH5 competent cells. This protocol describes preparation of the vector DNAs, analogous to that presented in Protocol 4 for preparation of the shuttle vector DNA by the two-step method. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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