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Fingerprinting Over Scotch Tape Shanna Bookal June 26, 2012 BSC 2010L ~ Biological Sciences I: Cellular Processes

Section 008 Lab Partner: Brenna Kaufmann

Materials and Methods The following is a comprehensive list of all of the materials that were used in our Structure of DNA Fingerprinting experiments. P20 Micropipette Graduated Cylinder Gloves Microtest tubes Marker (to label test tubes) Waterbath 250 mL Flask TBE Buffer Scale Plastic wrap Eletrophoresis Apparatus DNA samples Enzymes 1 &2 InstaStain Methylene Blue Agarose

In the methodology of this lab, it is pivotal to note that the appropriate lab attire should be worn at all times including, but not limited to, vented goggles, gloves and lab coat.

Preparation of the DNA Four microtest tubes were labeled 1-4 and had a group members initials placed on it in order to tell it apart from other lab groups. Then, using a micropipette, 10 L of Enzyme Reaction Buffer (Rxn Buffer) was dispensed into each of the four reaction tubes that were labeled. The Enzymes and DNA were also added to the test tubes. Table 1: Reaction Tube Reaction Buffer (L) Suspect 1 Suspect 2 1 2 3 4 10 10 10 10 DNA 1 (L) 15 15 0 0 DNA 2 (L) 0 0 15 15 15 0 15 0 0 15 0 15 Enzyme 1 (L) Enzyme 2 (L) Final Volume (L) 40 40 40 40

Once the Enzymes and DNA were added, all of the reaction test tubes were incubated in a 37 C waterbath for 45 minutes. Upon cooling after the incubation period was completed, 5L of 10x gel loading solution was utilized in order to prohibit the reaction of the enzymes and DNA. Further samples were stored in the refrigerator for later usage.

Electrophoresis Apparatus Set Up 0.40 grams of agarose was weighed out and mixed with a 50mL buffer solution in a flask in order to create a 0.8% gel solution once an electrophoresis apparatus was obtained. The flask was then covered over with plastic wrap in order to prevent evaporation during its 1 minute heating in the microwave to dissolve the agarose. Once the heating was finished and the agarose left to cool down, the gel tray was set up so that that the gel comb was at a certain end and the sides of the tray were facing positive and negative ends to make sure that the 0.8% solution would form into a square upon its addition to the tray. Upon solidification, the tray was removed and repositioned in order for the well rows to face the negative end of the electrophoresis apparatus. 250mL of 1X TBE Buffer was then added to the apparatus so that the surface of the agarose gel was submerged. Finally, the gel comb was removed from the agarose gel and the 30 L of each DNA sample could then be loaded.

Lane (Read from Left to Right) 1 2

Test Tube

Contents of Test Tubes

Markers CS1

Standard DNA Fragments DNA from crime scene with enzyme 1

CS2

DNA from crime scene cut with enzyme 2

DNA from suspect 1 cut with enzyme 1

DNA from suspect 1 cut with enzyme 2

DNA from suspect 2 cut with enzyme 1

DNA from suspect 2 cut with enzyme 2

Markers

Standard DNA Fragments

After the sample DNA was loaded and the apparatus was secure, the power was then turned on in which the black wire as connected to the negative pole and then red wire to the positive pole. Set at 150V and 150mA, the power was run for 40 minutes and upon the expiration of the time period, the power was cut off.

Gel Staining The agarose gel was stained with one sheet of InstaStain Methylene Blue by placing on the side with the well rows and pressing lightly upon it. At first, the gel did not show up very well so the TA added another sheet to it. After the staining, the gel was placed in a weighing tray and washed off with distilled water in order to remove excess gel so that

the DNA movement could be easier seen. The bands could now have the capability of being noticed although most groups bands did not show up very well or at all

RESULTS The results that were derived were not as intended. It was very tedious trying to make any distinctions between the features that were seen in the agarose gel. The end markers were also not very prominent and there was an appearance of a dark band within the gel. From the lack of quality in the gel, it is hard to make any precise results. However, as seen in figure 1 below (this is the universal image given to the class to analyze), it is seen that suspect 2 is the match with the crime scene DNA. The DNA matches multiple bands on the gel and lines up with crime scene 1.

Figure 1

Discussion It is clear that one can surmise that suspect 2s DNA matches with that of the DNA found at the crime scene. Nonetheless, this does not dictate that suspect 2 is guilty. The suspects DNA could have been there for any number of reasons such as a recent visit, contact with the victim prior to, within or after the crime, the suspect lives there, etc. Although the suspects DNA was located at the scene of the crime it does not indicate that they are guilty of the crime committed.

In this experiment there are a number of possible sources of error that could possibly cause invalidation of results. Unclean test tubes could affect the outcome of the experiment for other DNA would contaminate it. If there was inaccuracy in the measurements of enzymes, buffer or DNA this could skew the process of how the gel turns out. Also, loading solution was inaccurately added, the enzyme could potentially destroy the DNA. In this experiment, it was somewhat tedious trying to pipette the enzymes and DNA into the test tubes, especially when one is not accustomed to doing so. Secondly, a lot of the tubes did not contain the full 10L of reaction enzyme or 15L of DNA that was required. Also, in the pipetting of the DNA into the well rows, some rows overflowed into the solution which could potential cause band smearing.

If restriction enzyme 1 was the only enzyme that was utilized, then the experimental results would have been quite altered. Suspect 1 would have possibly been placed at the crime scene instead or alongside suspect 2. DNA fingerprinting is not used for criminal investigating but for other things such as maternity and paternity testing. DNA fingerprinting can also be utilized in the discovery of diseases, people identification and geographic mapping of inherited traits. This process has served to be most useful.