OT^'aaiflples of feces from mice iniecced by the oral route 
will be examined for the presence of polyoma virus. 
STAGE 3. The Preparation of Recombinant DNA Molecules Consisting of 
Polyoma and Col E 1 Components and their Subsequent Inoculation 
into Test Animals 
A. DNA 
1. Polyoma DNA will be purified and cleaved with R* Eco R1 as 
described in Stage 2, Al. 
2. Col El plasmid, or a derivative .thereof (obtained following 
consultation with Dr. Herbert Boyer, U.C., San Francisco) will be prepared 
from an appropriate strain of Eh, coli K-12. Purity of plasmid DNA will 
be assessed by agarose gel electrophoresis and cleavage with appropriate 
restriction endonucleases. 
B. Ligation and Transfection 
1. Oligomeric polyoma DNA will be prepared as described in 
Stage 2, Bl. 
2. Eco R1 cleaved Col El DNA, or a derivative thereof, will 
be ligated to oligomeric forms of polyoma DNA in the reaction mixture 
outlined in Stage 2, B2(a). 
3. A sample of the reaction mixture will be examined for 
evidence of ligation as described in Stage 2, B2(b). 
4. Transformation of E. coli will be effected as described by 
Cohen ct al. (PNAS 69, 2110-2114, 1972) in the presence of 0.003 M CaCl 2 
under P-4 conditions . Approximately twenty clones will be characterized. 
Xnitally, the clones will be screened by nucleic acid hybridization and 
subsequently by heteroduplex analysis and digestion of supercoiled DNA 
with restriction endonucleases. The bacterial clone containing the 
[Appendix C — 84] 
