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Example sentences for: spheroplasts

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The targeting hooks were placed in the promoter region of URA3 as shown in Figure 2. Genomic DNA and the linearized TAR cloning vector were combined with yeast spheroplasts and the His +Ura -transformants were selected on medium containing 5-FOA to identify clones with genomic DNA inserts.
The strain is a derivative of VL6-48 with the ura3-52 gene replaced by the KanMX cassette [ 15 ] . Spheroplasts were generated as described previously [ 29 ] . Agarose plugs (60 ul) containing approximately 2 ug of high molecular weight DNA were prepared from normal human fibroblasts MRC-5 (ATCC) or from liver cells of the Tg.AC mouse [ 11 ] . Linearized TAR cloning vector (1 ug) was added to the plugs, treated with agarase, and mixed with yeast spheroplasts.
The vector was linearized between the B1 targeting hook and URA3 and co-transformed with genomic DNA into yeast spheroplasts.
Optimum conditions for TAR cloning have been recently described [ 30 ] . Transformants were selected on synthetic histidine-minus plates containing 5-fluoroorotic acid (5-FOA) to select clones that no longer express URA3 [ 31 ] . Because of its toxic effect on yeast spheroplasts, 5-FOA was not included in the top agar.
Yield of the transformants varied from 100 to 300 colonies/ug vector, 2 ug genomic DNA, and 2 × 10 8spheroplasts (Table 3).