Question

RESULTS: Analyzing your data Answer the following questions: 1) How many bands do you see in the "unculanes? If there are more than one bad explain the nature or origin of the other bands? If there are more than one band can you 2) Why did you have to electrophorese uncut DNA? 3) What size of fragments were you expecting when you digested the known samples of pBR322 with the enzymes you used? 4) What are the sizes of the bands that you actually see? 5) Are there any other anomalous bands in these lanes? If so, can you explain how these may have arisen? 6) Does the plasmid you isolated correspond to pBR322? Use the photograph of your results to argue one way or the other. This is the central question, which should help support or negate our original hypothesis Page 31

Answer 1

1. Usually an uncut vector is circular which faces friction when subjected to electrophoresis which further results strain on the DNA molecule so that the conformation changes to twisted and knotted. Usually 2-3 bands can be seen in an uncut vector depending upon the conformations to which they belong i.e. linear, circular, open. These forms of uncut DNA move at different rates over electrophoresis gel with supercoiled moving at fastest rate. However, when it is subjected to restriction digestion, then the tension/strain is relieved resulting in the appearance of single band over the agarose gel.

2. Uncut DNA during cloning experiments act as a negative control so that we can differentiate if the transformation has happened successfully. In cut DNA two bands would be seen, one for vector and another for insert.