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One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of Plasmids.

TitleOne-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of Plasmids.
Publication TypeJournal Article
Year of Publication2020
AuthorsHeintz N, Gong S
JournalCold Spring Harb Protoc
Volume2020
Issue7
Pagination098095
Date Published2020 07 01
ISSN1559-6095
Abstract

In the one-step approach to bacterial artificial chromosome (BAC) modification, two plasmids are introduced into the BAC host cells. The shuttle pLD53.SC2, carrying the EFGP reporter sequence and requiring the π protein to replicate, must be grown in PIR1- or PIR2-competent 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 30°C; here we use DH5α competent cells. This protocol describes preparation of the vector DNAs. The shuttle-reporter vector DNA is subsequently digested for introduction of one homology arm (typically the A-box).

DOI10.1101/pdb.prot098095
Alternate JournalCold Spring Harb Protoc
PubMed ID32611776