A standard alkaline-lysis method is effective for purifying TAC plasmid DNA of ~100 kb from either E. coli or Agrobacterium. The plasmid DNA can also be purified rapidly and cleanly with the commercially available plasmid preparation kit QIAGEN-tip 20. For plasmid preparation from E. coli, we add 7 ml of LB medium containing 0.7 mM IPTG to 3 ml of an overnight culture of transformed E. coli, and then shake the culture for 5 h at 37C before plasmid preparation. Several micrograms of DNA are usually obtained. From Agrobacterium, we obtain 1 microgram of DNA from 6 ml of cells cultured for 24 h.
A TAC plasmid containing a DNA insert of ~100 kb can be introduced efficiently into Agrobacterium strains by electroporation according to the method of Mattanovich et al.(Nucleic Acids Res.17, 6747, 1988). We use 10 to 100 ng of TAC plasmid DNA per 40 ml of competent cells. If colonies of various sizes are apparent on the agar plate, clones of each size should be picked for plasmid preparation. Our experiments suggest that larger colonies usually harbor plasmids with deleted inserts. We recommend preparing plasmid DNA from several colonies, even if they appear to be of the same size. Given that the digestion patterns of plasmids isolated from A. tumefaciens are not clear, we also recommend retransferring plasmids into E. coli DH10B and then comparing the Hind III digestion patterns between the original and reisolated plasmids from E. coli for confirmation of the integrity of plasmids in A. tumefaciens.

Protocol 2. Southern blot analysis of I-SceI digests of genomic DNA

Megabase fragments of DNA isolated from transgenic plants (20 g) are embedded in low-melting point agarose plugs (Liu, Y.-G. and Whittier, R.F. Nucleic Acids Res.22, 2168-2169, 1994). The agarose blocks are cut to a size of ~4 by 4 mm (~30 mg) and washed with 0.2x TE buffer in 1.5-ml tubes. To each tube are added 6 microL of 10x digestion buffer without Mg2+, 20 microL of distilled, deionized water, 1 microL of I-Sce I enhancer, and 20 to 40 U of I-Sce I (Boehringer Mannheim). The tubes are placed on ice for 100 min, after which the reaction in initiated by the addition of MgCl2 to 5 mM. After incubation for 60 min at 37C, the reaction is terminated by the addition of 1 ml of TE buffer. The digestion products are separated on a 0.8% agarose gel by pulsed-field gel electrophoresis (Bio-Rad) and subjected to ultraviolet irradiation (5 x 100000 J) with a UV Stratalinker 2400 (Stratagene). The DNA molecules are then transferred to a nylon membrane by the alkaline method, and the blot is hybridized with an HPT gene probe.

Protocol 3. Preparation of an Arabidopsis genomic DNA library with the TAC vector

Megabase fragments of A. thaliana genomic DNA were isolated by the method of Liu and Whittier (Nucleic Acids Res.22, 2168-2169, 1994), and partially digested with Hind III for ligation into the TAC vector. The isolation, partial digestion, fractionation, and concentration of genomic DNA are described by Liu, Y-G. et al. (Plant J. 7, 351-358, 1995) . The vector pYLTAC7 was digested with Hind III and ligated with the Arabidopsis genomic DNA overnight at 16C. The resulting plasmids were then introduced into E. coli DH10B (Gibco BRL) by electroporation. Transformants were selected on LB plates containing kanamycin (25 microgram/ml) and sucrose (5%), and were stored in 27 384-well plates at -80C.