1.A.2 Continued

One example of an evolutionary change that is partially related to a change in the environment is the emergence of dog breeds over hundreds of years. It has been impacted by both environmental as well as human factors. From the environmental standpoint, the regions from which certain breeds emerged played some part into what physical characteristics canines adapted. For example, the dense, thick fur of the Siberian Husky allowed it to endure the extremely cold and harsh climates of the Siberian Arctic. The Mexican Hairless Dog is a breed whose lack of hair allows it to be comfortable in the Central American heat. The role of humans has played a very large impact on the evolutionary change in these animals. When we bred ancient dogs for certain tasks, they developed characteristics suited to those tasks over many, many generations. For example, the muscular, compact body of the Portuguese Water Dog allowed it to herd fish into nets, retrieve tackle, and serve as courier from boat to land. Our actions in this respect were artificial selection. While dog breeds will continue to change and emerge, similar developments may be seen in similar species in the future. Domestication of foxes in recent years has notably resulted in foxes with dog-like traits such as less erect tails.

Essential Knowledge 1.A.2 (3D GameLab)

Peppered Moth Simulation

21.
 Light Forest -> 77% light moths and 23% dark moths at end
 Dark Forest ->  33% light moths and 67% dark moths at end

22.
In a light forest, a light-colored moth would have a much greater chance at survival because it can blend easily into its surroundings. The tan color of the bark was the same as if not similar to the color of the light-colored moths in that simulation. A light-colored moth would be in stark contrast to the light background, and predators would be able to pick it out much more quickly. In the same way, in the dark forest, a dark-colored moth would be much harder for a predator to find than a light-colored moth that would stick out so easily from its surroundings.

23.
Natural selection dictates that in a situation with variation in traits, differential reproduction, and heredity, the phenotype that allows the organisms that best chance at survival will continue to exist and be carried on in the next generation. In this example, the light or dark color that allows the greatest chance at survival from predators in the light or dark environment, respectively, will be passed on by the moths of that color to the offspring.

24.
If there were no predators in this case, the colors of the moths might still change, though not nearly as dramatically. Moths of both light and dark colors would continue to proliferate. The colors within the moth population might only change depending on the nature of the colors' genotype; if the relationship between the dark and light genes was incomplete dominance, there may be an intermediate color (perhaps gray) that would appear.

Restriction Mapping of DNA Plasmid Lab

Restriction Mapping of DNA Plasmid Lab

Purpose

This lab explores the use of restriction mapping by the use of restriction enzymes followed by gel electrophoresis in characterizing a DNA sequence. Restriction enzymes are used to digest DNA samples, and because each restriction enzyme is known to correspond to a certain sequence, their use gives clues as to what nucleotides are at those sites. They are also used in the creation of recombinant DNA; the needed sequence is isolated using restriction enzymes before insertion into a vector and connection with DNA ligase. The band lengths of DNA that are produced by the restriction enzymes and measured by means of electrophoresis can help determine the location of restriction sites to create a map of the given DNA.

Introduction

First, agarose gel is cast, liquid to gel with 6 wells to pipet the DNA into it. Once there is DNA in the wells it can go through electrophoresis, which uses the negative charges of DNA molecules to pull fragments through the gel. The wells are at the negative side of the chamber, and as the current runs through the chamber the dye and fragments will move through the gel toward the positive electrode. The speed of the DNA fragments’ movement is determined by their relative sizes (bigger fragments move slower), and since the electrophoresis is stopped before any of the bigger fragments can catch up to the smaller ones, the smaller fragments will move further through the gel.

The lambda/PstI DNA is what the other sites are being compared to. The others are single, double, and triple digests, which space out along the gel.

After the electricity moves through the gel and the bands move the gel is removed and one can compare the marker DNA to the other DNA in the chambers.

Methods

In the procedure of this lab, it should be noted that many steps were already performed by the teacher and teacher assistant in the casting of the agarose gel and setup of the electrophoresis chamber. As a lab group, we need merely load the samples into their respective wells.

The entirety of a sample would be drawn into a fresh pipet. It was made sure that the sample was expelled of air so air bubbles would not form around the well. The pipet would then be steadied over the well with two hands, deep enough to touch the bottom of the well and without pressure that would puncture the gel itself. The sample was then slowly injected into the well. This was done with lambda and the four DNA samples. The order of our samples was pMAP/PstI, pMAP/PstI/SspI, pMAP/PstI/HpaI, and pMAP/PsI/HpaI/SspI.

If we believed additional sample was needed for successful electrophoresis, more was injected into the well. The second well in our gel was also left empty.

Above: the gel before electrophoresis and the samples next to their corresponding wells

Above: loading a DNA sample into its well

The gel was carefully placed in the electrophoresis chamber and left until the longest DNA fragment was about 2 cm above the end of the gel.

Below: the gel in the electrophoresis chamber

Above: several gels in the electrophoresis chamber

After electrophoresis, the gel was taken by the teacher for staining.

Below: gels in the process of being dyed for analysis

Above: the gel in the middle of electrophoresis

Data

Above: the gel after electrophoresis

Above: the gel on the light box with and without an orange filter

Below: marked DNA fragments on the lightbox

Below: Estimations of the band lengths from the electrophoresis

Graphs & Charts

Graphs and charts were not necessary to the analysis of this lab. However, the lab document included these maps of the DNA fragments when cut with alternate combinations of restriction enzymes.

Discussion

Using the lambda/PstI lane for comparisons, we were able to approximate the lengths of the bands in each of the test lanes. The DNA cut with PstI only (lane 3) showed two resulting bands of lengths approximately 2838 bp and 900 bp; with PstI and HpaI together (lane 4), it showed three bands of lengths 2000, 1800, and 1000 bp; with PstI and SspI (lane 5), it showed three bands of lengths 2500, 900, and 650 bp; with all three (lane 6), it showed four bands of lengths 2000, 1200, 900, and 650 bp. The average total DNA length for each lane turned out to be 4335 bp, which we rounded to 4500 for simplicity’s sake. Using this, we converted the individual band lengths into usable estimations that added up to the rounded average for our plasmid map: the bands of lane 3 became 3500 and 1000 bp; lane 4’s became 2000, 1500, and 1000 bp; lane 5’s became 2700, 1000, and 800 bp; and lane 6’s became 1500, 1200, 1000, and 800 bp. Using these approximated lengths we determined the relative restriction sites on the plasmid as shown below.

Above the map, we have the original band lengths in black with reappearing band lengths highlighted in the color of the restriction enzyme associated with them, as well as the adjusted estimations in orange.

Conclusion

Due to rounding errors in the analysis of the gel, estimates of band lengths and restriction sites were not completely accurate. The appearance of the bands in the gel was also not completely clear, which contributed to errors in band measurement and plasmid mapping. The DNA cut with PstI only in particular had a total DNA length that was pretty far from the average (3738 bp vs 4335 bp), so that required some significant rounding. Despite all of the rounding and estimating, however, we have determined the relative locations of the restriction sites for the three enzymes with some level of accuracy.

References

The procedures and figures used were obtained from the document “Restriction Mapping of Plasmid DNA” from the Carolina Biological Supply Company.