40 

 plasmid DNA, the pellets were first dissolved in sterile 

 water, then 4M NaCl and 13% PEGgooo was added, followed by 

 incubation on ice before centrifugation at 4°C. The pellets 

 were rinsed in ethanol, dried under vacuum and resuspended 

 in sterile water. DNA concentrations were determined 

 spectrophotometrically at 260 nm and by agarose gel 

 electrophoresis with a known DNA standard. Method 2: The 10 

 ml cultures, as prepared above after centrifugation, were 

 treated with prepared solutions provided by Promega 

 (Madison, WI) in the Magic Miniprep DNA purification kit. 

 The DNA was passed through a mini -column and dissolved in 

 GTE (Promega instructions for preparation) buffer. This 

 rapid method is preferred for sequencing on a Li -Cor 

 sequencer available in the Department of Microbiology and 

 Cell Science. 

 Competent cells of E. coli strains 



E. coli strains DHBo;, AB1360 {aroD) and SK494 [aroE) 

 were grown overnight in 50 -ml tubes and inoculated into 1-L 

 flasks containing 200 ml LB medium. The cells were 

 incubated at 37°C in a shaker at 3 00 rpm for several hours 

 until a spectrophotometric reading at Agog nm between 0.4 and 

 0.5 was reached. Cells were harvested by centrifugation in 

 sterile tubes after incubation on ice, treated with 100 mM 

 CaClz as described by Sambrook (68) , resuspended into 100 mM 

 CaClj with 15% glycerol, aliquoted into microcentrifuge 

 tubes, dropped into liquid nitrogen and stored at -70°C 



