Use of differential agar media for detection of cloned DNA fragments in the tetracycline and chloramphenicol resistance genes of pBR322.
A method for detecting newly cloned DNA fragments in pBR322-based vectors was devised for use in DNA probe production. Escherichia coli strain DH5 containing plasmids with different resistance patterns to tetracycline (Tc) and chloramphenicol (Cm) were grown on nonpigmented media, blotted, transferred, and incubated for 2 h on MacConkey agar containing Tc or Cm. Resistant colonies changed color to pink as they began fermenting the lactose on the agar, while sensitive colonies remained white but were still viable and could be subcultured. This method can be applied to the detection of other plasmids with insertional inactivation of Tc or Cm resistance marker genes following successful cloning experiments, especially if pUC18 or M13 is not a possible vector. It eliminates 1 day of culture and the labor involved in individually transferring hundreds of colonies.
Thornton
S A
SA
U.S. Naval Medical Research Unit No. 3, Cairo, Egypt.
D002702:Chloramphenicol Resistance / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D010957:Plasmids; D013753:Tetracycline Resistance / Q000235:genetics*
D003470:Culture Media; D004269:DNA, Bacterial; D004274:DNA, Recombinant