Plant Molecular Biology Reporter pages 82-94 13 (1) 1995 Protocol Isolation of Megabase-Size DNA from Sorghum and Applications for Physical Mapping and Bacterial and Yeast Artificial Chromosome Library Construction Sung-Sick Woo, Vipin K. Rastogi, 1 Hong-Bin Zhang, Andrew H. Paterson, Keith F. Schertz and Rod A. Wing 2 E-mail: rodwing@tam2000.tamu.edu (S-SW, VKR, H-BZ, AHP, RAW) Department of Soil and Crop Sciences, (KFS) USDA-ARS, Texas A&M University, College Station, TX 77843, USA Key Words: Sorghum bicolor, megabase-size DNA, YAC and BAC cloning, physical mapping Abstract: A method was developed for the isolation of megabase-size DNA from Sorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than I Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries. T he recent developments of PFGE technology (Schwartz and Can- tor, 1984; Chu et al., 1986) and cloning systems for large DNA fragments, such as YACs (Burke et al., 1987) and BACs (Shizuya et al., 1992), has led to valuable information about complex genomes of 1Present address: Department of Biochemistry and Biophysics, Texas A&M University. 2Corresponding author. Abbreviations: BAC, bacterial artificial chromosome; CHEF, clamped homoge- neous electric field; CIAP, calf intestine alkaline phosphatase; LMP, low melting point; LMW, low molecular weight; PFGE, pulsed-field gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SPD, spermidine; YAC, yeast artificial chromosome. 82