gene disruption

[Mercy]

Hi,
  I need some advise on this one. 
  Recently i  modified a certain S. cerevisae yeast vector by gene disruption (of leucine) to include zeocine antibiotic as a selection marker. Leucine gene was cloned, ligated, and finally transfromed into DH5 alpha before disruption. I confirmed the disruption by several methods and made a glycerol stock (YPD broth + zeocin, and 25% glycerol) of the recombinant yeast. Now, i want to isolate the yeast vector for ligation. I have tried liquid culturing the glycerol stock into LB broth and it doesnt grow. Any ideas and/or pieces of advise will be highly appreciated. 

 		
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