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GENE CLONING I. Questions and Experimental Observations 1. First examine and compare the growth of bacteria that received no DNA when plated to a plate containing no ampicillin to the plate that contained ampicillin. What is the phenotype of the original E. coli culture BEFORE transformation? In this experiment if they didn't take up a plasmid, were they ampicillin sensitive? 2. Count and record the number of colonies on the plates that were incubated with ’uncut’ DNA and ’cut’ DNA. If you have thousands of transformants, you can divide the plate into four or six sections. Count the number of transformants in one section and multiply by the number of sections to get a pretty good estimate. Type of DNA | Cut plasmid DNA | Uncut plasmid DNA Number of Colonies (White Light) | | Number of Colonies (UV Light) | | 3. The cut DNA represents linear DNA, while the uncut represents a circular plasmid. How does the transformation efficiency of the linear DNA compare to that of the circular plasmid? Can E. coli be transformed equally well with any form of DNA? 4. The growth on the ’No ampicillin’ No DNA plate gives an indication of the total number of cells plated. The colonies on the uncut DNA indicate the number of competent cells (those that took up the ampicillin resistance plasmid). Comparison of these two plates gives an indication of the percentage of the total bacteria that take up plasmid. If you didn't have ampicillin selection to help in identifying the bacteria that took up the plasmid, would they be easy to find? 5. What new phenotypes have been acquired by the transformed bacteria (those that take up the plasmid)? How have their appearance and ability to grow been changed?

GENE CLONING
I. Questions and Experimental Observations
1. First examine and compare the growth of bacteria that received no DNA when plated to a plate containing no ampicillin to the plate that contained ampicillin. What is the phenotype of the original E. coli culture BEFORE transformation? In this experiment if they didn't take up a plasmid, were they ampicillin sensitive?
2. Count and record the number of colonies on the plates that were incubated with ’uncut’ DNA and ’cut’ DNA. If you have thousands of transformants, you can divide the plate into four or six sections. Count the number of transformants in one section and multiply by the number of sections to get a pretty good estimate.
Type of DNA | Cut plasmid DNA | Uncut plasmid DNA
Number of Colonies (White Light) | |
Number of Colonies (UV Light) | |
3. The cut DNA represents linear DNA, while the uncut represents a circular plasmid. How does the transformation efficiency of the linear DNA compare to that of the circular plasmid? Can E. coli be transformed equally well with any form of DNA?
4. The growth on the ’No ampicillin’ No DNA plate gives an indication of the total number of cells plated. The colonies on the uncut DNA indicate the number of competent cells (those that took up the ampicillin resistance plasmid). Comparison of these two plates gives an indication of the percentage of the total bacteria that take up plasmid. If you didn't have ampicillin selection to help in identifying the bacteria that took up the plasmid, would they be easy to find?
5. What new phenotypes have been acquired by the transformed bacteria (those that take up the plasmid)? How have their appearance and ability to grow been changed?

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