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Two-Step Bacterial Artificial Chromosome (BAC) Engineering: Verification of Co-Integrates and Selection of Resolved BAC Clones.

TitleTwo-Step Bacterial Artificial Chromosome (BAC) Engineering: Verification of Co-Integrates and Selection of Resolved BAC Clones.
Publication TypeJournal Article
Year of Publication2020
AuthorsHeintz N, Gong S
JournalCold Spring Harb Protoc
Volume2020
Issue4
Pagination098087
Date Published2020 04 01
ISSN1559-6095
Abstract

Successful modification of the bacterial artificial chromosome (BAC) after two-step BAC engineering is confirmed in two separate polymerase chain reactions (PCRs). The first reaction (5' co-integrate PCR) uses a forward 5' co-integrate primer (a sequence located upstream of the 5' end of the A-box) and a reverse 3' primer on the vector (175PA+50AT) or within the reporter sequence or mutated region as appropriate. The second reaction (3' co-integrate PCR) uses a forward 5' primer on the gene (RecA1300S) and a reverse 3' co-integrate primer (a sequence located downstream from the 3' end of the B-box). Those colonies shown to be positive in PCR analysis are further tested for sensitivity to UV light. After the resolution, colonies that have lost the excised recombination vector including and genes become UV light sensitive.

DOI10.1101/pdb.prot098087
Alternate JournalCold Spring Harb Protoc
PubMed ID32238594