42 

 Subcloninq cDNA for complete sequence analysis 



Subcloning of cDNA inserted into pBluescript was 

 performed by use of appropriate restriction enzymes to 

 obtain fragments for sequencing both strands of the entire 

 cDNA. Plasmids used for fragment insertion {pUC18, 

 pGEM5zf(+), or pBluescript SK+} were incubated with the same 

 restriction enzyme (s), and were treated with calf intestinal 

 alkaline phosphatase (CIAP) (when only one RE was used) to 

 prevent rejoining of ends during ligation. Cloned cDNA 

 fragment samples and plasmid samples were loaded onto 1% DNA 

 agarose gels at 70V for about 1 h. Fragments and linear 

 plasmid DNA were excised and treated with the Gene Clean 

 system (Bio 101, LaJolla, CA) , prior to ligation and 

 transformation . 

 Li-Cor sequencincT of subcloned cDNA fragments 



Plasmid DNA was denatured and reacted with forward or 

 reverse fluorescent primers, enzymes, 7-Deaza dGTP sequence 

 extending mix and NTPS according to the directions supplied 

 by Li-Cor (Lincoln, NE) . Three /il of each of four prepared 

 sample mixtures containing one of the four dideoxy 

 nucleotides was loaded onto wells of a urea polyacrylamide 

 gel set into the Li-Cor model 4000L sequencer. Parameters 

 were set in a computerized data collection file and the 

 sequencer was run overnight . The sequences were analyzed in 

 a data analysis computerized system. 



