The involvement of genes 3, 4, 5 and 6 in genetic recombination in bacteriophage T7

Genetic Recombination of Bacteriophage T7 Dna in Vitro

“…5. The ratio of the ss-, amber+ recombinant phage to the total (4,5), are also required for in vitro recombination.…”

“…T7 diluent consisted of 0.02 M Tris-HCl buffer, pH 7.4, 10 mM MgSO4, 0.5% NaCl, and 10 ttg/ml of gelatin (1,14). CA medium contained per liter 2.0 g of vitamin-free casamino acids, 5.0 g of NaCl, 1.0 g of NH4Cl, 12.0 g of Tris base. The pH was adjusted to 7.4 with HC1 and after autoclaving 2 ml of 20% glucose was added per liter of medium.…”

“…coli BL2 [iig-, no suppressor activity (Su0)] was from F. W. Studier (12). Shigella sonnei D2 571-48 (Su0) and a Su+ derivative (Shigella sonnei [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] were obtained from R. Hausmann (13 7.4 with NaOH. The concentrations of tryptone and yeast extract were doubled to make 2 X LB.…”

“…To obtain 32P-, density-labeled phage, overnight cultures of E. coli B or BBW/1 grown in CA medium were diluted 50-fold into 250 ml of CA medium and grown at 300 to a cell density of about 1 X 108 per ml (OD65o = 0.08). FdUrd and BrdUrd were added to final concentrations of 10 ,ug/ml and 200 jig/ml, respectively, and H332P04 was added at a concentration of 5 ,uCi/ml. Shaking was continued for 3.5 hr, by which time cell growth ceased (OD65o = 0.35).…”

“…Many of the gene products which have been implicated in T7 recombination in vivo are available in purified form (4)(5)(6)(7)(8)(9).…”