Title | One-Step Bacterial Artificial Chromosome (BAC) Modification: Preparation of Plasmids. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Heintz N, Gong S |
Journal | Cold Spring Harb Protoc |
Volume | 2020 |
Issue | 7 |
Pagination | 098095 |
Date Published | 2020 07 01 |
ISSN | 1559-6095 |
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). |
DOI | 10.1101/pdb.prot098095 |
Alternate Journal | Cold Spring Harb Protoc |
PubMed ID | 32611776 |