Molecular Cloning oHarge DNA Fragments of The Salt Tolerant wild Tetraploid bermudagrass Cynodon Dactylon L. Using A Bacteriophage Cloning Vector. 2. Ligation and in vitro packaging of The Recombinant Phage DNA Molecules

This paper represent the second step in a molecular cloning program aiming to clone large D N A fragments of the salt tolerant bermudagrass ( Cynodon Jadylon L.) D N A using the bacteriophage (EMBL3) as a vector. In this work, a yield of about 100 j.ig bacteriophage D N A per one liter culture was obtained with.a purity ranging between (1.7-1.8). The vector DNA was completely double digested with the restriction enzymes BamHl and FcoRl, followed by purification of vector arms. Bacteriophage arms were also efficiently ligated with the partially digested bermudagrass DNA (prepared earlier). The optimum ligation ratio of arms: inserts (EMBL3: bermudagrass) was found to be 4:1 respectively. The recombinant DNA was successfully in vitro packaged and plated on a P2 lysogen E. coli (strain NM539). An efficiency of about 1.3xl0