1 f iUA- lb 
- 10 - 
B: Non- Infectious Material for Biochemical Analysis 
1. Lysate of bacteria containing P-plasmid DNA recombinant molecules. 
a. Cultures containing P-plasmid recombinant DNA will be shaken 
with 1% chloroform at room temperature for ten minutes. 
b. One milliliter of the treated culture will be added to L broth 
containing diamimopimel ic acid and thymidine and incubated at 
37 C for 24 hours. 
c. If the medium remains sterile, the preparations will be removed 
from the Class III cabinet: 
(I) As described in Section I.2.(C).l, Page 18, of the Safety 
and Operations Manual for work conducted in the Mobile 
Containment Laboratory; 
- or - 
(ii) Through a chemical dunk tank and then surface-decontaminated 
with ethylene oxide gas prior to removal from Building 550, 
FCRC. 
2. Lysates of bacteria containing P-lambda DNA recombinant molecules. 
a. In those casts where P-lambda lysates will be examined for the 
presence of polyoma-specific RNA or polypeptides, £. col i K12 
strain supO (non-suppressor containing host) will be used. 
Under these conditions, no progeny phage of P-lambda Warn 403 
Lam 1100 Sam 100 will be made. The bacterial cultures will be 
treated with 1% chloroform and assayed for the presence of 
viable bacteria as described above. 
b. Materials derived from experiments in which X phage vectors are 
employed will be tested also for the absence of P-X phage using 
JE. col i DP-50 supF. Materials can be considered free of P-X 
phage if 20yl of the undiluted lysate or serum yield no plaques 
when plated with E. col i DP-50 supF. 
c. If the L broth remains sterile and no plaques are seen, the 
preparation will be removed from the Class III cabinet and the 
P4 facility by procedures described in 1 X ( B ) 1 Ci or IX(B)lCii 
above. 
3. P-lambda DNA 
a. P-lambda DNA will be prepared from purified P-lambda phage 
particles by deproteini zati on with SDS-phenol under P4 conditions 
b. The bacterial sterility of purified DNA will be assessed by the 
addition of 10yq of DNA to L broth followed by a 24-hour 
incubation at 37C. 
[Appendix C — 240] 
