Construction of bacterial artificial chromosome (BAC/PAC) libraries.
This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.
Osoegawa
K
K
Roswell Park Cancer Institute, Buffalo, New York, USA.
de Jong
P J
PJ
Frengen
E
E
Ioannou
P A
PA
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D005826:Genetics, Medical; D015698:Genomic Library; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D008970:Molecular Weight
D004247:DNA