This plasmid has two antibiotic resistance genes, Amp^R and Kan^R. Amp^R and Kan

Question

This plasmid has two antibiotic resistance genes, Amp^R and Kan^R. Amp^R and Kan^R make E. coli cells resistant to antibiotic Ampicillin and Kanamycin, respectively. It also has three restriction enzyme sites, EcoRI, BamHI, and HinDIII. See the figure to the right. What restriction enzyme do you use to clone DNA fragments digested by EcoRl into this plasmid? What enzyme do you use to create recombinant plasmids with a human DNA fragment by mixing the plasmid and DNA fragments prepared above? In order to find E. coli cells transformed with the recombinant plasmids prepared above, what antibiotic do you add to culture media plates? When you try to create these recombinant plasmids, a success rate is not 100%. In other words, many of them do not have any DNA fragment. How can you identify E. coli cells such "empty" plasmids? What antibiotic do you use to find recombinant plasmids that have a DNA fragment at the HinDIII site? List all possible antibiotics.

Explanation / Answer

ANSWER:

1. Restricted enzyme EcoR1 is used to clone the DNA fragments into this plasmid because if use same enzyme to cut both plasmid DNA and cloned DNA framents ,same sticky ends will be produced in both DNA.

2.DNA Ligase is used to create recombinant DNA by mixing both framents.

3.As cloned DNA is inserted by using EcoR1 enzyme , kanamycine resistant gene will be inactivated which is so called insertional inactivation . so inorder to find the transformants kanamycine antibiotic can be used .

4.while culturing the Ecoli cells on kanamycine antibiotic medium , the cells which lost the resistance against that antibiotic are transformants or recombinants and those which retain resistance are non transformants or nonrecombinants.

5.As none of the antibiotic gene is located at the HinDIII enzyme for given plasmid, we cannot use any antibiotic for selection.

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