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Cloning Based on Efficient Three-Fragment Assembly,,,DNA Ligation

d by Dr. John McCafferty (Cambridge Antibody Technology).
Two DNA fragments (here designated as fragments A and B) had to be joined at a common unique Alu I site and ligated with a fd-tet DNA backbone at Apa LI and Bam HI restriction sites (Figure 1). Both insert fragments were obtained by cleavage of cloned DNA with the endonucleases Apa LI, Bam HI, and Alu I. The Apa LI/Alu I fragment of insert A and the Alu I/Bam HI fragment of insert B were purified after separation on an agarose gel. One microgram of DNA in a total volume of 10 l was used in the conventional ligation reactions, with a molar ratio of phage DNA to insert A and insert B of 1:5:5. Standard ligations of the two insert fragments together with appropriately cleaved fd-tet DNA were performed for 24 h at 14 C. Our optimized procedure was comprised of two temperature steps: first, the blunt-end ligation of the insert fragments A and B for 18 h at 26C; second, the cohesiveended vector DNA was added, and the ligation continued for an additional 6 h at 14C [3]. The protocol for the Rapid DNA Ligation Kit was modified in accordance with our optimized conventional procedure. The blunt-end ligation mixture (290 ng DNA in a total volume of 20 l) was ligated for 30 min at 26C. Next, 710 ng vector DNA was added in a volume of 1 l with further incubation for 10 min at room temperature.

The ligation mixtures were then cooled to 0C without heat-inactivation of the ligase, and one third of each reaction was used to transform DH5a competent cells, which were plated on 140 mm Petri dishes containing 30 mg tetracycline/ml LB medium. After a 20 h incubation period, colonies were counted, and doublestranded phage DNA of a representative number purified with the High PureTM Plasmid Isolation Kit. The DNA was screened by restriction analysis with the
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