38 

 Preparation of cDNA libraries for screening 



The cDNA library was first titered, then mixed with the 

 freshly prepared host strain, XLl-Blue, plated with top agar 

 on about 80 LB plates at about 20,000 PFU (about 1.6 X 10® 

 PFU/ desired clone) and incubated at SV^C. Isopropylthio-/8- 

 D-galactoside treated nitrocellulose filters were applied 

 about 6 to 8 h later and incubation was continued overnight. 

 Filters were removed and the immunological screening 

 procedure of Sambrook et al . (68) was followed. 

 Screening the cDNA library of Lambda ZAP II 



Nitrocellulose filters removed from the plates were 

 washed, soaked in blocking buffer containing dry milk, 

 rocked gently in primary S-protein, ADH or PAT specific 

 antibody/blocking buffer and washed several times. The 

 filters were next exposed to secondary antibody, goat anti- 

 rabbit antiserum, washed as before and incubated with 

 substrates, nitroblue tetrazolium, 5-bromo-4-chloro-3- 

 indolyl phosphate and with 5 mAf MgClz. The filters were 

 kept in the dark and gently rocked for one to two hours. 

 Plaques corresponding to the small, deep blue spots on the 

 filters were cored from plates, dispensed into SM buffer 

 plus chloroform and stored at 4°C for titering and plaque 

 purification. 



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