|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|
|Date Published||2020 07 01|
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).
|Alternate Journal||Cold Spring Harb Protoc|