Jason
Trejo
Jordan
Purpose: The purpose of this lab was to run DNA through the process of gel electrophoresis using 3 restriction enzymes in order to characterize DNA. We were then meant to prepare and analyze our results based on the data we procured by running the DNA through the gel
Introduction: Restriction mapping is used to determine the size of DNA fragments. In gel electrophoresis, the smallest fragments will always travel a larger distance and the bigger fragments will travel a shorter distance. The negatively charged end of the gel electrophorese repels the DNA moving it towards the positive end. Restriction maps of DNA are the equivalent to fingerprints, put alongside Lambda DNA as a size comparison and control, we were able to characterize the alien DNA.
Methods:
First of all we obtained a gel from our teacher and then we delicately proceeded to load the tiny wells with the contents of each reaction tube, containing DNA.
(The prepared enzyme/DNA solutions and the Agarose gel.)
With my super technique I was able to skillfully use a needlepoint pipet to load the wells of the gel, being careful that the pipet is void of any air bubbles that could spill the solution.
(Agarose gel after DNA has been administered.)
(Agarose gel being cast into the electrophoresis tank)
The negatively charged phosphate backbone of the DNA allows for it to move through the pores of the heated Agarose gel, towards the positively charged anode at the opposite end of the wells. The lighter fragments move farther because they can squeeze through pores more easily and are faster due to their lighter weight.
The completed gel electrophoresis under an orange lamp that allows us to have a better look at where the fragments ended up on the gel. We used sharpie on a plastic Baggie to emphasize the cut points and label the respective enzyme/DNA solutions for easier reference. Cut sites had to be estimated, so the numbers were not adding up to be equal, however the concept and relative lengths of each fragment was understood and noted.
Data:
Discussion: Through gel electrophoresis we were able to obtain enough data to map the plasmid and discovered that the DNA sequence was cut by the restriction enzymes on several occasions. There were two PstI sites , one SspI site, and one HpaI site on the plasmid. Compared to the Lambda DNA, this mystery DNA had much shorter fragments when PstI was administered.
Conclusion: Although our cut sites and fragment lengths did not necessarily add up correctly, we were still able to insert estimated numbers and create a plasmid displaying the data we procured. We were able to determine the enzyme cut sites alone, and in relation to each other during the double and triple digests. The smaller bands move through the gel more quickly due to their size, so we were able to make educated guesses on the positions of cut sites with given number data.
