-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
Transformation initially defined as horizontal gene transfer method of plasmid but later on it became widely accepted molecular biology method. In this process competent cell, logarithmically or exponentially dividing cell (freeze in cryo-protected media), will intake foreign plasmid DNA (hydrophilic in nature) by using chemical method (in presence of CaCl2) or electric shock method (electroporation). These cells further use for long term storage of DNA (cloning host genetically lack of recombinase A and endonuclease A deficient; recA-, end A-) or expression of foreign protein in bacterial cells.
Transformation include following steps:
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