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United States Department of Agriculture

Agricultural Research Service

Title: High Throughput Bac DNA Isolation for Physical Map Construction of Sorghum (Sorghum Bicolor)

Authors
item Klein, Robert
item Morishige, Daryl - TEXAS A&M UNIV
item Klein, Patricia - TEXAS A&M UNIV
item Dong, Jianmin - TEXAS A&M UNIV
item Mullet, John - TEXAS A&M UNIV

Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 1, 1998
Publication Date: N/A

Interpretive Summary: The next major advancement in plant genetics hinges on the development of physical maps. With a physical map, genes controlling important agronomic traits can be localized, isolated and eventually manipulated to aid in crop improvement. One approach to producing a complete physical map attempts to clone the entire genome and then to determine the order in which the clones swere present in the target genome. One of the first steps of constructing a physical map of cereal grains is to isolate cloned pieces of the plant genome that have been inserted into a bacterial artificial chromosome. This study details protocols that permit the isolation of large numbers of recombinant bacterial artificial chromosomes containing large fragments of the sorghum genome. These protocols are necessary for the construction of a physical map of cereal grains. This information will be primarily used by fellow scientists.

Technical Abstract: With an aim of constructing a physical map of sorghum, we developed a rapid, high throughput approach for isolating bacterial artificial chromosome DNA suitable for restriction endonuclease digestion fingerprinting, polymerase chain reaction-based content mapping, and bacterial artificial chromosome-end sequencing. The system utilizes a programmable 96-channel liquid handling system and associated accessories that permit bacterial cultivation and DNA isolation in 96 well plate format. The protocol details culture conditions that optimize bacterial growth in deep well plates and criteria for bacterial artificial chromosome DNA isolation to obtain high yields of quality DNA. The system is robust, accurate, and relatively cost effective. The bacterial artificial chromosome DNA isolation system has been tested during efforts to construct a physical map of sorghum.

Last Modified: 9/22/2014
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