39 

 Excision of Pbluescript from the Lambda ZAP II vector 



Purified phage stocks were incubated with E. coli 

 strain XLl-Blue and ExAssist helper phage to excise the 

 pBluescript plasmid from the Lambda ZAP II vector for 2 to 

 2.5 h at 37°C in 2XYT media (see Stratagene instruction 

 manual for details) , heated to 70°C and centrifuged. The 

 supernatant, containing the plasmid, was incubated with host 

 E. coli strain SOLR, spread on LB plates containing 50 mg 

 ml"^ ampicillin and incubated overnight at 3 7°C. Colonies 

 from these plates were streaked on fresh LB-AMP plates for 

 immediate use and were also stored in DMSO for future use. 

 DNA purification 



Plasmid DNA was purified by two methods. Method 1: The 

 pBluescript plasmids containing putative cDNA clones coding 

 for the S-protein, ADH or PAT were purified for sequencing 

 by the method of the DNA Sequencing Core Facility, ICBR, 

 University of FL. A colony from the E. coli SOLR strain 

 from the LB -AMP plate above was incubated overnight in 10 ml 

 of LB-AMP medium in a 50 ml tube for each clone sample. The 

 cultures were centrifuged and the pellets were resuspended 

 in GTE (68) buffer. Treatment with NaOH/SDS was followed by 

 neutralization with potassium acetate at pH 4.0, and 

 centrifugation was carried out to remove cellular debris. 

 The supernatant s were treated with DNase-free RNase and 

 extracted twice with chloroform. DNA was precipitated with 

 isopropanol and washed with 70% ethanol . To precipitate the 



