Why is the DNA fragment amplified with Phusion DNA into pGEM-T plasmids? The colorless E. coli colonies in our experiment contain the recombinant pGEM-T/GFPuv plasmid. Some of the colonies emit green fluorescence whereas others don\'t when they are irradiated with long wave UV. light. Explain Why. Solution 1) Phusion DNA Polymerase enzyme is a proof-reading polymerase, with extreme specificity. The enzyme provides robust amplification. Since it is a proof-reading enzyme, Phusion DNA Polymerase creates blunt ends in the DNA fragment, because of which it cannot be directly cloned into the plasmid. 2) GFPuv ,the green fluorescent protein, exhibits structural polymorphism. Onlyproper conformationproduces fluorescence. Some colonies emitted fluorescence, because the GFPuv in them is in proper conformation while in colonies which did not emit fluorescence, the protein was not in a proper conformation..

1. Why is the DNA fragment amplified with Phusion DNA into pGEM-T plasmids? The colorless
E. coli colonies in our experiment contain the recombinant pGEM-T/GFPuv plasmid. Some of
the colonies emit green fluorescence whereas others don't when they are irradiated with long
wave UV. light. Explain Why.
Solution
1) Phusion DNA Polymerase enzyme is a proof-reading polymerase, with extreme specificity.
The enzyme provides robust amplification. Since it is a proof-reading enzyme, Phusion DNA
Polymerase creates blunt ends in the DNA fragment, because of which it cannot be directly
cloned into the plasmid.
2) GFPuv ,the green fluorescent protein, exhibits structural polymorphism. Onlyproper
conformationproduces fluorescence. Some colonies emitted fluorescence, because the GFPuv in
them is in proper conformation while in colonies which did not emit fluorescence, the protein
was not in a proper conformation.