2. Lambdaphage 
Xgt-XB will be obtained from Dr. Ron Davis, Stanford 
University and propagated *as described by Thomas £t £l . (PNAS 71, 4579- 
4583, 1975) in E. coli strain C600. Phage DNA will be purified as 
described by Thomas and Davis (J. Mol. Biol. 91^, 315-329, 1975) and 
cleaved, initially analytically, and then preparatively, with R* Eco Rl. 
B. Ligation and Transfection 
1. Preparation of oligomeric polyoma DNA 
a) Eco RI linear molecules of polyoma DNA (50 ugm/ml) 
will be incubated with T4 ligase in the presence of 0.1 M Tris-HCl, pH 
7.9, 50 mM NaCl, 1.0 mM EDTA, 12 mM MgClj, 10 mM DTT, 0.1 mM ATP and 0.1 
ug/ml bovine serum albumin at 10°C. 
b) A sample of the reaction mixture will be analyzed by 
electrophoresis through 0.7Z agarose. If suitable amounts of oligomeric 
polyoma DNA have been generated, the entire reaction mixture will be 
subjected to preparative agarose gel electrophoresis and dimeric through 
tetrameric polyoma DNA bands eluted from the gels. 
2. Preparation of polyoma-bacteriophage lambda recombinant 
DNA molecules 
a) Eco Rl cleaved lambda DNA (50 yg/ml) will be briefly 
heated to dissociate terminal sequences and incubated with oligomeric 
forms of polyoma DNA (50 pg/ml) in 0.1M Tris-HCl, pH 7.9, 50 mM NaCl, 
1.0 mM EDTA, 12 mM MgCl 2> 10 mM DTT, 0.1 mM ATP, 0.1 pg/ml bovine serum 
albumin and T4 ligase at 10°C. 
b) A sample of the reaction mixture will be checked for 
evidence of ligation by electrophoresis in 0.7% agarose, restriction 
enzyme cleavage patterns and electron microscopic analysis of heteroduplexes. 
[Appendix C — 82] 
