Purpose: The purpose of this lab is to analyze unknown DNA to determine the number of cut sites for each restriction enzyme and the positions of those cut sites relative to one another. The goal was to use electrophoresis to help us visualize the placement of each of the bands compared to one of the master bands in order to find out the size of each.
Introduction: Restriction mapping is a critical step in analyzing and characterizing a new DNA sequence. A restriction map of a piece of DNA can be compared to a fingerprint, something to identify each piece. In order to set up this lab, we were to use three restriction enzymes, PstI, HpaI, and SspI. The purpose of this lab, as stated before, is to find out the number of cut sites present in the DNA sequence for each restriction enzyme and the positions of those cut sites relative to one another. In order to do this, the DNA should be loaded into each well of the gel and analyzed after gel electrophoresis has taken place.
Methods: Fortunately, the agarose gel was already pre-made when we did our experiment, so we did not have to worry about casting the agarose gel. Our first task was to simply load the DNA into the gel. We began by drawing samples into the pipet, making sure we expelled any excess air so that air bubbles wouldn’t form in the wells. We steadied the pipet over the gel and dipped the pipet through the surface of the buffer, positioning it over the well, and letting out the liquid. We did this a total of 4 times, one for the comparison or control group, and the rest for each of the three different restriction enzymes. The next task was the actual electrophoresing process. This consisted of an electrophoresis chamber which was connected to a power supply with high voltage. Our teacher monitored the electrophoresis and took it out when he saw fit and then we loaded it onto the staining tray. After being stained, we were given back our trays to be able to examine the movement of each restriction enzyme bands through a lightbox. The lightbox helped see the bands better so that we could label them and compare by size marker.
Discussion: The movement of the different bands in each well were dependent on their varying sizes. The well that showed the farthest movement down the gel towards its positive end was well Pst I. This can lead us to assume that the DNA strands that were present in that solution had strands that were cut thin enough so as to be able to make its way so far down it's given well. In contrast to that, the well of Pst I also had a marker in it that did not travel far in comparison to the rest, meaning that it's DNA strands were much too large to move much distance at all. When compared to the constant well that was the Marker well, most other wells only conducted of a given 2-3 bands. This would lead us to assume that the DNA we were working the wells with we're relatively basic in the sense that not many bands were present or shown. ThBeing pre-given approximations for the positions where each marker should have ended, accuracies in their placement verified that the correct loading substance was placed into its appropriate well. Furthermore, after constructing the positions of the various recorded bands into a visual circular chart for every well and it's bands, the lengths in which they were recorded straight on the gel came to match with the circular graphs,
Data:
Conclusion: Through the use of gel electrophoresis, we were able to identify where certain cuts in all restriction enzymes occurred. We were able to label the bands we saw under the lightbox and compared it with the control well in order to find out our efficiency. After taking a close look, we realized not all of the bands that were supposed to be present were visible. This may have been due to an error in loading the wells. We realized after the put away our try to the electrophoresis station that one of our tubes had not been fully pipetted into the well and that may have caused some inaccuracies in our results. We acknowledged our mistakes and realized it could have been avoided by making sure we expelled all excess air from out pipet. However, aside from small mistakes, our data was pretty accurate in reflecting what should have been seen according to our data. Wells 2-4 were all supposed to have the same total size of DNA fragments and as seen in our data, it was within the same range, since it was an estimate of course. As different fragments of DNA moved at different rates and therefore different positions, we used this data to construct diagrams of said fragments.
