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Title: You have just inserted a recombinant plasmid into a bacterial cell? Post by: irina on Oct 3, 2012 You have just inserted a recombinant plasmid into a bacterial cell. The cloning vector originally contained a tetracycline resistant gene and the LacZ gene. To determine whether or not the bacterial contains the recombinant plasmid you grow the bacteria on media. What MUST the media contain?
Title: Re: You have just inserted a recombinant plasmid into a bacterial cell? Post by: kh006 on Oct 4, 2012 The medium must first contain the antibiotic tetracycline ,in this way all the bacterial cells that have been transformed by the uptake of the cloning vector and thus antibiotic resistant will survive while the remaining cells die. next you should distinguish between the cells that carry a plasmid cloning vector with a DNA insert from the ones carrying a plasmid cloning vector without one ( there is a possibility that during the procedure of preparation of the cloning vector that cut plasmid might reanneal before the dna fragment to be cloned is inserted)
To do this the medium must contain the organic compound x-gal which once acted upon by the enzyme encoded by the lac Z gene gives a blue color as a result you should screen for white colonies because they are the ones that have a disrupted lac Z gene (due to the presence of the dna insert) |