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High Throughput BAC DNA Isolation for Physical Map Construction of Sorghum (Sorghum bicolor)
Authors:Klein  Robert R.  Morishige  Daryl T.  Klein  Patricia E.  Dong  Jianmin  Mullet  John E.
Affiliation:(1) Southern Crops Research Laboratory, USDA-ARS, 2765 F& B Road, College Station, TX 77845, USA;(2) Department of Biochemistry-Biophysics and Crop Biotechnology Center, Texas A&M University, College Station, TX 77843, USA
Abstract:
With the aim of constructing a physical map of sorghum, we developed a rapid, high throughput approach for isolating BAC DNA suitable for restriction endonuclease digestion fingerprinting, PCR- based STS-content mapping, and BAC-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. This protocol details culture conditions that optimize bacterial growth in deep-well plates and criteria for BAC DNA isolation to obtain high yields of quality BAC DNA. The system is robust, accurate, and relatively cost-effective. The BAC DNA isolation system has been tested during efforts to construct a physical map of sorghum.
Keywords:BAC  BAC-end sequencing  DNA isolation  sorghum
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