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Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box).

TitleTwo-Step Bacterial Artificial Chromosome (BAC) Engineering: Preparation of the A Homology Arm (A-Box) and B Homology Arm (B-Box).
Publication TypeJournal Article
Year of Publication2020
AuthorsHeintz N, Gong S
JournalCold Spring Harb Protoc
Volume2020
Issue4
Pagination098046
Date Published2020 04 01
ISSN1559-6095
Abstract

The 700-bp A homology arm (A-box) and the 700-bp B homology arm (B-box) are amplified by polymerase chain reaction (PCR) using purified bacterial artificial chromosome (BAC) DNA as template for two-step BAC engineering. The resulting A-box PCR product contains an AscI site at its 5' end (the 5' primer incorporates an AscI site, and the 3' primer does not incorporate any restriction sites). The B-box PCR product contains an XmaI site at its 3' end (the 5' primer does not incorporate any restriction sites, and the 3' primer incorporates an XmaI site). The amplification products are then digested with the appropriate restriction endonucleases to render them suitable for cloning into the shuttle vector.

DOI10.1101/pdb.prot098046
Alternate JournalCold Spring Harb Protoc
PubMed ID32238590