Construction and screening of BAC libraries made from Brachypodium genomic DNA

Kerrie Farrar; Iain S. Donnison

doi:10.1038/nprot.2007.204

Construction and screening of BAC libraries made from Brachypodium genomic DNA

Kerrie Farrar, Iain S. Donnison

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

47 Citations (Scopus)

Abstract

Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.

Original language	English
Pages (from-to)	1661-1674
Number of pages	14
Journal	Nature Protocols
Volume	2
Issue number	7
DOIs	https://doi.org/10.1038/nprot.2007.204
Publication status	Published - 28 Jun 2007

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title = "Construction and screening of BAC libraries made from Brachypodium genomic DNA",

abstract = "Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.",

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N2 - Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.

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Construction and screening of BAC libraries made from Brachypodium genomic DNA

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