An ampicillin-resistant, tetracycline-resistant plasmid is cleaved with EcoRI, w

Question

An ampicillin-resistant, tetracycline-resistant plasmid is cleaved with EcoRI, which cuts within the tetracycline gene. The cut plasmid is ligated with EcoRI-digested Drosophila DNA to prepare a genomic library. This DNA is then used to transform E. coli K12. A) Under what conditions should you grow bacteria in order to select for transformed cells (those that have taken up a plasmid)? B) How can you identify bacteria carrying a plasmid that contains a fragment of the Drosophila DNA? C) How can you explain the presence of colonies that are resistant to both antibiotics?

Explanation / Answer

A) The bacteria should be grown under the media containing ampicillin. As the bacterial cells that have taken up the plasmid develop a resistance to ampicillin whereas the other cells do not, due to lack of ampicillin resistant gene from plasmid.

B) The bacteria which contain plasmids having the fragment of Drosophila DNA are resistant only to ampicillin not tetracyclin. This is because the tetracyclin gene is cleaved to insert the drosophila DNA and hence is non-functional and cannot code for tetracyclin resistance.

C) When the plasmids cleaved with EcoRI the sticky ends produced are complimentary to each other. And when these ends are being ligated with EcoRI cleaved Drosophila DNA, there are fare chances that the sticky end of plasmid re-ligate to itself without incorporating the drosophila DNA. Thus, keeping its tetracyclin resistant gene intact and the thus formed colonies are resistant to both antibiotics.

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