Light Blue Lab Report

Draw lines to represent where the bands will be.

6,400
5140
4600
4310
4240
4160

3820
2880
2520
2500

2000

*light blue is 1 band

Experiment The group first designed the materials to create and hold the .75 agarose gel and experiment. Everything except the comb was made of ¼ inch Acrylic Plexiglass. The glass helped the group see what was going on in the experiment. The comb was 3D printed to be more accurate. One material was a casting tray which held the Gel in future steps. The casting tray had to hold at least 50 mL of liquid. They didn’t want the casting tray to hold much more than 50mL or else they’d be wasting materials. The group sealed two sides of the casting tray to the base with Bostik All Weather Sealant. The
sides they glued on had indents for the comb later. The other two sides of the casting tray were taped on for then. The casting trays base was 5.6 cm by 2 cm, and 2 cm tall. Next, the comb was created with teeth long enough to create wells in the .75 agarose gel. The comb had 5 teeth to create 5 wells, and the neck of the comb was wide enough to sit on the sealed walls of the casting tray. The comb was 6cm wide, with 1.4 cm long teeth. The wells the comb created were used later as a holder for DNA. The chamber was created to hold 200mL with two holes in one side. Each side was sealed together. Electric plugs were stuck through the holes that way electricity could later be run through the chamber. One plug was negative, and one was positive because DNA is negatively charged, and will move towards the positively charged current. The lid was then made to fit over the chamber, and had two holes on the side which matched up to the holes on the chamber. That way, when the lid was pulled off the electricity was also disconnected for safety. The lid was a 9.2 by 2.0 cm piece attached on the edge to a 7.6 by 9.2 cm piece. Last the group made the chamber. The chamber had to be big enough to hold 150 mL of buffer solution and the casting tray. They made the base of the chamber 9.2 by 6cm, and 7.6 cm high. The group then created the Gel by mixture.
The .75 agarose gel is used so scientists can see when DNA runs through it. They mixed 45 mL of the TAE buffer with .75 grams of agarose powder. The group used TAE buffer rather than water because the TAE buffer is a salt based liquid. It contains a mixture of Tris base, acetic acid and EDTA. This means it is positively charged, and when electricity is run, the TAE buffer guides the electricity from negative to positive, which will be crucial for a future step. This happens because two valence electrons release, and move towards the positive charge. The group then boiled the mixture and then let it cool to 80 degrees Fahrenheit. The group then poured the mixture into the casting tray making sure the teeth of the comb reached the .75 agarose gel. Once the .75 agarose gel hardened, the group put it in the fridge overnight. Then, they took the taped sides off the .75 agarose gel. This is so electric current could run through the .75 agarose gel without being stopped by the wall. The group also took the comb out, that way there were wells to insert DNA
later. The group next mixed the DNA samples with Restriction Enzymes. The DNA samples collected were crime scene DNA, suspect one’s, suspect two’s, and suspect three’s DNA. The DNA was purchased from BIO-RAD. Each one had 10 microliters placed in separate microtubes. The group then added 10 microliters of Restriction Enzyme into each of the four microtubes. The Restriction Enzymes cut the DNA into different sections. This is a crucial part of the experiment because once the DNA is cut, the different sections of DNA will be different lengths. Bigger sections will move less in the .75 agarose gel, and the group will be able to compare each DNA fingerprint once the experiment ran. The group then centrifuged the tube for 30 seconds, and incubated the tubes for 24 hours. Centrifugation “separates cell debris from DNA-containing supernatant,” (Gray, 2016) that way the DNA can be extracted. Next the group added wires to the negative and positive plugs. The wires are what carried the electric currents through the experiment. The wires went down and then ran parallel to the chambers base, but never touched any of the glass. Once the DNA had incubated for 24 hours, the group added 5 microliters of loading dye into each microtube. The loading dye made the DNA more visible, and brought the DNA down to the bottom of the well. The group then centrifuged the tubes for 30 seconds. The group added 150 mL of buffer to the chamber so electric currents could run negative to positive. Then, the group took the .75 agarose gel out of the casting tray because it didn’t fit very well into the chamber. They made sure the side of the .75 agarose gel with the wells was closer to the negative charged current. This way, once the DNA was inserted and electricity was run, the DNA would repel from the negative charge, and attract to the positive charge.
The group then pipetted into the wells. Starting with the well 2nd to the left the group added the crime scene DNA. Next, the group added suspect one, suspect two, and suspect three from left to right in separate wells. They then added the Hind III ladder to the leftmost well. The ladder was used to compare the results, to know how many bp each band was.. The group made sure the wire was not touching the .75 agarose gel, or else it would melt. Next the group plugged in the power and ran the experiment at 75 volts for 20 minutes. Because the .75 agarose gel was short, the group did not run 150 volts, or the DNA would go off of the .75 agarose gel. By running the electric current the DNA which is negatively charged was able to attract towards the positive side of the .75 agarose gel. This is the step where restriction enzymes play their role. If one section of DNA is bigger than another due to the cutting of the restriction enzymes, it will take more time to move through the .75 agarose gel towards the positive charge. By comparing different bands where the sections of DNA ended up, the group was able to compare each DNA sample. The group then removed the .75 agarose gel from the chamber, and wrapped it in plastic wrap. They placed it in the fridge to stay solid.
The next day the group dyed the .75 agarose gel. They placed the .75 agarose gel in a dye bin for 10 minutes, and then a rinse pan for 5 minutes to remove excess dye. The group did this step once more until the .75 agarose gel was very blue. The dye was used so the group could see the bands of DNA. The group took pictures of the bands, and observed and compared each band. Results

Analysis The .75 agarose gel electrophoresis experiment is used to compare different DNA fingerprints due to Restriction Enzymes, and negative and positive charges.
Each DNA fingerprint should have several bands. The ladder would have been used as known DNA lengths to compare the DNA of the crime scene and suspects. The groups experiment did not go completely smoothly for a few major reasons. To begin with, each DNA fingerprint had no more than one band. This made it impossible to figure out who committed the crime because there was no available comparison to the crime scene DNA. Next, suspect two had no visible DNA. This major error made it impossible to observe the DNA fingerprint of that suspect. This caused the group to have a missing sample, and crucial information.
Conclusion The initial hypothesis for suspect A was that the bands would be at 6,400, 5140, 4600, 4310, 4240, 4160, 3820, 2880, 2520, and 2500 base pairs. The group's gel ended up only showing one band each. There also was no bands for the groups suspect two. The factory image on the other hand, showed three bands for each suspect so the group decided to continue using that. The group decided to identify solutions to the problems they faced. A possible solution to only having one band would be to change the agarose to TAE buffer ratio. The group put in 45 mL of buffer rather than 50 because the casting tray was smaller than average. This caused the ratio of agarose to TAE buffer to be wrong. When agarose mixes with buffer, and is added to heat and dissolved, one cooled the mixture turns into a gel. When there is more agarose the gel is thicker. Due to this inaccurate proportion, the .75 agarose gel ended up thicker than hoped for. The DNA had trouble moving through this thick .75 agarose gel, because when a substance is denser, it takes more energy to move through it. Another possible reason that there was only one band is because the casting tray was too short. Rather than putting the wells on the long side, the group should have put the wells on the short side. This gives the DNA more room to run, and not as many bands would have run off the .75 agarose gel. If the .75 agarose gel was longer, the group could also run more voltage (150 volts), and the .75 agarose gel would attract to the positive charge faster. The smaller strands of DNA move through the gel faster, and because the groups gel was so short the smaller strands ran right off.The group concluded that to observe more bands, the .75 agarose gel needed to be longer on the side opposite of the wells so the DNA could move towards the positive without going out of the .75 agarose gel. Another problem that occurred was suspect two had no visible DNA. This problem was caused by user error during pipetting. When the group added suspect two’s DNA into the microtube, pressure on the pipette was released before it was pulled out of the tube. By doing this, the pipette picked up the DNA in the microtube rather than releasing it. Because there was no DNA, there was no visible representation of it. The group did not get the correct results, so instead, they looked at the factory image. The factory image was the same experiment, except with correct results. When looking at the factory image, because the crime scene DNA bands sizes (bp) were 2,200, 2,000, and 1,800, and suspect three’s bands were 2,200, 2,200, 1,900bp. they concluded suspect three was guilty. They had the closest correlating bp. Suspect one’s bands were 2,000, 1,970, and 1,900 bp and suspect two’s bands were 2,000, 2,050, and 2,050 bp. In the groups experiment, they should have concluded the same results, but they made major errors. What should have happened is the group got those bp measurements, but because of the errors the group made in the experiment, they got zero to one band for each DNA tested. Plenty of value came of this to me. When I learned about DNA fingerprinting, and performed this lab, it really made me interested in the medical field. I found it more enjoyable to learn about enzymes because I understood them, and more importantly a real world example of how the enzyme can play roles in fingerprinting. For example learning how the ECOR1 enzyme broke up DNA, and how that can apply to DNA fingerprinting was very fascinating. By researching a little bit more about enzymes, I learned plenty of applications I could use in my profession. For example, “pancreatic enzymes have been in use since the nineteenth century for the treatment of digestive disorders” (Chaplin, 2014). I have learned there are plenty of therapeutic roles enzymes have. When I grow up I may want to become a doctor, and by learning about different roles enzymes have in the body, I have gotten one step closer to that career option. One huge piece of information I learned from the experiment is the importance of organization and teamwork. If it wasn’t for a great team, the experiment would not have gotten done, and if the group was not organized we wouldn’t know which step each person was on.