Problems in plasmid prep..

VASANTHARAJAN JANAKIRAMAN vasant at cc.UManitoba.CA
Wed Sep 10 08:18:36 EST 1997


Hi everyone,
I had constructed a cDNA lib. using stratagene's lambda -zap kit, I did 
about 3 screenings and did manage to get some positives. Inorder to 
recover the plasmid from the phagemid I did the excision and finally 
plated the cells on LB+amp plates. I used a little portion of the 
bacterial colony (on the LB+amp plate) to do PCR amplification and teh 
remaining portion I inoculated in LB+amp broth o/n, in microfuge tubes 
and on the subsequent day (I did observe growth) I froze the tubes in 
-70"c, with 20%  glycerol. 
My amplification worked, I even did a dot blot with my PCR products and 
they indeed were positives. But when I wanted to do a plasmid prep and 
streaked a loopful onto LB+amp plates (they grew very well on the 
plates) and inoculated a single colony from the plate into 5 ml LB+amp 
broth, there was no growth. My plates were fresh, I had even prepared 
fresh LB broth. I even called the technical support at Stratagene but no 
one seems to know the source of the problem. I would like some 
suggestions as to what could gone wrong.
thanks in advance
vasanth  



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