Monday, November 3, 2008

Q: Explain how a foreign DNA fragment can be introduced into a plasmid that produces blunt ends.

Cut the DNA fragment and plasmid using the same restriction enzyme. Add guanines to DNA fragment blunt ends and terminal transferase, to produce a single chain of guanines to both the blunt ends of the plasmid vector. Add cytosines to vector blunt ends, to produce a single chain of cytosines to both blunt ends of the DNA fragment.
Mix the cut plasmid and cut DNA fragment together for recombination to occur. Single chain guanine will bind to single chain cytosine by complementary base pairing. Add ATP and DNA ligase to seal the nicks by forming phosphodiester bonds in the sugar backbone.
Add recombinant DNA to bacteria culture for transformation to take place. Use heat shock and add calcium chloride to facilitate uptake of recombinant DNA.

From here on, answer is question specific...
Culture bacteria in agar plate containing ampicillin (assuming original plasmid had ampicillin resistant gene). Bacteria that has taken up the plasmid vector, whether reannealed or with the foreign DNA will be resistant to ampicillin and grow on the agar plate. ...

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