-You have three separate plasmids in your lab, but the labels have come off of t
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Question
-You have three separate plasmids in your lab, but the labels have come off of the tubes and you no longer know which plasmid is which. For the time being, you will refer to the plasmids are plasmids 1, 2, and 3. You know the following:
-One plasmid has kanamycin-resistant gene.
-Two plasmids have an ampicillin-resistance gene.
-In addition to having an antibiotic-resistance marker, one plasmid also codes for the gene for green fluorescent protein (GFP), a protein from the bioluminescent jellyfish Aequorea victoria. Bacteria that produce the green fluorescent protein look green under white light and fluoresce under ultraviolet light.
-Your PI has tasked you with determining the identity of the antibiotic resistance gene in the plasmid containing GFP. Write a detailed protocol describing exactly how you would do this. Include all steps of the transformation making sure to include all the controls you will need. Your protocol should be clear enough for a brand new student in your lab to follow.
Explanation / Answer
It is a relatively easy experiment.
Take two aliquots of each plasmid and transform competent cells. Plate them separately on both Ampicillin and Kanamycin plates separately. Label the plates carefully (ex: Plate 1: Plasmid 1 + Ampicillin).
So, we will have total six plates. Incubate them overnight at 37'C and observe for the colony growth next morning. Next morning, we can observe colonies only on three plates.
Take these plates and write down the antibiotic resistance marker present in each plasmid. Then, expose these plates to UV light and observe for fluorescence. Only one of the three plates exhibits fluorescence. Mark that plate and report to your boss which antibiotic marker is present along with the GFP in which plasmid.
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