Restriction Mapping Lab

Purpose

     In this experiment, we separated DNA fragments by the size of their base pairs and analyzed the digested sites where restriction enzymes cut. This helped determine the number of cut sites for each restriction enzyme and their positions next to each other. We determined the total size of the DNA strands by adding up the sizes of the fragments from each digest. Since we knew that smaller DNA fragments migrate quicker than the larger ones, we were able to use the data for gene mapping.

Intro

     Molecular biology techniques often include the use of restriction enzymes to digest DNA as well as the separation of DNA fragments with the help of agarose gel electrophoresis. These techniques can be used for gene mapping and even for studying human genetic diseases. Restriction enzymes are enzymes that have the property to catalyze the cleavage of certain DNA molecules at specific base sequences. They are used for chromosomal mapping and also for gene splicing in recombinant DNA technology. Gel electrophoresis is a method used in labs to separate DNA, RNA, and proteins by their molecular size. In the process, the molecules are separated by being pressed through a gel (often agarose gel) by an electrical field. A negative charge is applied so that the molecules move towards the positive charge to be analyzed. 

Methods

The first thing that we did was use a needle point pipet in order to load the DNA into the gels, the first column contained the pMAP or lambda (“clear”) which will act as a marker within our results. Skipping the second, the third column contained PstI (“blue”), the fourth PstI/SspI (“red”), the fifth PstI/HpaI (“white”), and the sixth column with all three PstI/SspI/HpaI (“yellow”).

A picture of our group's gel; GO GO GO!

After placing the DNA into the wells (which was actually harder than it looks… at least for our lab group…), we closed the top of the electrophoresis chamber and turned on the voltage. As a result, the DNA should move from one end to the other positive side of the electrophoresis apparatus.

When we came back the next day, the gels were stained in order to make it easier to see the marks on the gel.

Graphs and Charts

Our picture of a plasmid with the restriction enzymes in their approximate location. 

Discussion

Although our results were not the best because our group had trouble placing the DNA into the gel, we were still able to draw data from the results of other lab groups. With the help of the marker lane on the far left of the gel, we were able to approximate the distance between the different restriction enzymes within a plasmid. The PstI well should only have one marker, the middle should have two and the farthest right should have three markers. By using this approximation, the distance of the strands should have been a total of 3900 and each column should have that total.  

Conclusion
The results from the gel tell us the approximate length of each band, which allowed us to visualize and the draw a full plasmid, annotated with the locations of each band along the ring-like structure. This lab is unique because we see how biology comes into play with forensic science. Comparing the band patterns of a suspect and victim creates a very powerful convition case if there is a clear match. Gels are simple to use, easy to understand and remarkably accurate, so it's no wonder that this concept has appeared on the AP Biology test for 15 consecutive years.

References
https://barnard.edu/sites/default/files/inline/restriction_enzyme_digestion_lab.pdf

http://www.nature.com/scitable/definition/gel-electrophoresis-286