pamp, pkan, or pblu? Introduction In my final biotechnology lab project, I was given an unidentified plasmid, in which I was required to perform restriction digests using restriction enzymes. A plasmid, however, is a genetic structure in a cell that has the ability to replicate independently of chromosomes. Usually, plasmids are used to analyze DNA, as well as to create proteins. A restriction enzyme is an enzyme that is used to cut and isolate DNA. In order to review the results of our digest, however, I needed to perform a gel electrophoresis. This process allows you to insert your DNA samples into open wells in the gel, and separate the DNA fragments. Then, the gel would be placed into the gel electrophoresis chamber, where the samples will be run using an electrode, with the total distance traveled by the samples ranging between 4-5 centimeters. The goal of this lab was that I would learn how to perform a restriction digests, as well as to understand how to use a gel electrophoresis to separate fragments of DNA of a plasmid. This goal is important because these procedures would help me determine my unidentified plasmid, whether it was pamp, pkan, or pblu, as the title of this report suggests. In order to accomplish this, however, I needed to prepare three solutions into three 1.5 ml microtubes each with an equal amount of the plasmid: the control, the single digest, and the double digest, with the control containing no restriction enzyme. Once these solutions were prepared, I would digest all three of the tubes at 37 degrees Celsius for one hour. Meanwhile, I needed to prepare the gel, so that I could insert the DNA samples into it. However, before adding the samples to the gel, I needed to add 4 microliters of loading dye to the samples so that the DNA fragments would be visible for the photograph. I would then run the gel in the gel electrophoresis chamber to find the sizes of my base pairs. I would then take a picture of the gel, and would later determine the base pair sizes by making a standard curve.
Methods Prior to beginning this lab, I performed virtual restriction digests for pamp, pkan, and pblu using the restriction enzymes that I chose on the New England Biolabs website 1 in order to predict my digest results. In order to perform the virtual digests, I gathered the base pair sizes for all 3 plasmids from DNA Learning Center 2. For the double digest, I used the double digest section of the New England Biolabs website 3, in which I tested different combinations of enzymes, which is where I formulated my double digest recipe. If both enzymes could both be digested at the same temperature for the same amount of time, then the combination would work. Once I had performed the virtual digests, I used those numbers as the expected base pair sizes for data analysis. When I was given my plasmid, I found that it had a code of 6410F14. When I began my lab, my lab instructor told me that the starting concentration was 150 ng/ml. When I performed the gel electrophoresis, I used a marker DNA ladder that I would refer to for measurements in order to make my standard curve after the lab. For my restriction enzymes, I used BglI for the single digest, and BamHI & NdeI for the double digest. Then, for the reaction buffer, I used Buffer 3.1. The DNA ladder, the restriction enzymes, and the reaction buffer, all came from New England Bio Labs 1. For my digestion recipe, I calculated that for the single digest for BglI, I would need to digest in a heat block at 37 degrees Celsius for one hour. For my double digest using BamHI & NdeI, I would also need to digest them in a heat block at 37 degrees for an hour. While I waited for an hour, I needed to make my gel for the gel electrophoresis portion of the experiment. I used a 0.8% agarose solution, because we would be focusing on 12,000-8,000 base pairs for the gel. I then needed to measure 0.4 grams of the agarose solution into a flask, then I had to add 5mL of the 10X TAE solution that I prepared earlier, then I needed to bring the volume of the solution to 50 ml using dh 2O. Then, I placed the solution in a microwave, and heated it for about 2-3 minutes in a microwave. Afterward, I added 1 microliter of ethidium bromide to the solution. I poured the solution into a gel gasket, where it would solidify. However, shortly after I poured the solution into the gasket, I would insert a 10-tooth comb into
the gel so that I could insert my samples into the gel. Once the gel was prepared and ready to run after I inserted the solutions into the wells. Then, I had to cover the gel, and fill the box, with buffer, in which I did so with a TAE buffer. A month prior to working on this lab, I prepared a 10X TAE Buffer using Tris, Glacial Acidic Acid, and EDTA. A 10X TAE Buffer contains 0.4 moles of Tris with a formula weight of 121.14 g/mol, 0.2 moles of Glacial Acidic Acid with a formula weight of 17.5 M stock solution, and 0.01 moles of EDTA with a formula weight of 292.24 g/mol. However, we would only make 250 ml out of 1 L of the Buffer. So, in order to get the proper amounts for the buffer that we would use, I multiplied the moles of each part of the buffer by its molarity, and then multiply it by 0.25, which is the amount of each solution for the buffer. I ended up with 12.114 g of Tris, 2.86 ml of Glacial Acidic Acid, and 0.7306 g of EDTA. Once we mixed these, we measured the ph level of the buffer using a ph meter, in which we found that the ph level was 8.35. In this lab, I used 28 ml of the 10X TAE buffer, and then brought the solution to volume 280 ml using about 252 ml of dh 2O. I had to do dilute this in order to make my 1X TAE Buffer. I ran the gel at 140 volts, for approximately 40-45 minutes. When the DNA solutions ran past the 4 cm mark of the gel tray, I stopped running the gel, and removed it from the gel electrophoresis chamber. Then, I placed it in the UV transilluminator and photographed the gel. To discover the results, I created a standard curve on Microsoft Office Excel. First, I measured the distance traveled by the ladder fragments from the wells of the gel. Next, I recorded the distance, and used the numbers to create a chart with an exponential line, in which I found the equation for the line, which is what I used to help me determine the sizes of my DNA fragments. Then, I measured the distance traveled by all of my DNA fragments from all three samples from the wells of the gel. I plugged in the distance for one fragment in millimeters for x into the equation, in which I would calculate the base pair size of that fragment. The fragment sizes and other data for this lab is in the section below. Results and Conclusions Figure 1
Ladder Size (bp) Figure 1 is the image of the gel. Lanes 1: DNA Ladder. Lane 3: Control. Lane 5: Single Digest (BglI). Lane 7: Double Digest (BamHI & NdeI). Lane 9: DNA Ladder Figure 2 Unknown Plasmid y = 105640e -0.131x R² = 0.9916 12000 10000 8000 6000 4000 2000 0 0 5 10 15 20 25 30 35 40 Band migration (mm)
Figure 2 shows the standard curve for the gel. The x-axis shows the measured distance traveled of each DNA fragment for the DNA Ladder in millimeters, while the y-axis represents the number of base pairs for the DNA Ladder. The equation at the top right hand corner is the equation for the standard curve that was used to determine the base pair sizes for each fragment of the digests. The R 2 value shows that this graph is reliable because the number is close to 1, in which 1 represents a perfect line. Figure 3 Band Migration Ladder Size (bp) (mm) Ladder Band 1 19 10000 Ladder Band 2 20 8000 Ladder Band 3 21.8 6000 Ladder Band 4 23 5000 Ladder Band 5 24.7 4000 Ladder Band 6 26.5 3000 Ladder Band 7 30.2 2000 Ladder Band 8 32.5 1500 Ladder Band 9 36.4 1000 Control Band 1 12.4 20814 Control Band 2 19.8 7895 Control Band 3 24.8 4101 BglI 1 12.8 19752 BglI 2 19.1 8653
BglI 3 23.5 4614 BglI 4 25 3995 BglI 5 34.9 1092 B & N 1 12.3 21089 B & N 2 19.5 8212 B & N 3 25 3995 Figure 3 shows the distances traveled by the digests as well as the base pair sizes for each fragment. I used the equation from Figure 2 to plug in the distance traveled for a fragment for x to find the size of the fragment. Figure 4 BglI (Single Digest) BamHI & Nde I (Double Digest) pamp 3263, 1118, 158 3205, 1334 pkan 3139, 794, 261 3959, 235 pblu 2121, 1740, 1576 2976, 2461 Figure 4 shows the expected base pair sizes for pamp, pkan, and pblu for both the single and double digests. As I mentioned earlier in this report, my lab instructor informed me that the initial concentration was 150 ng/ml, and the final concentration would be 250 ng/ml. Figure 1, which is the image of the gel, shows the fragments of each digest. Figure 2 shows the standard curve, along with the equation to
determine the base pair sizes of the digests, in which I would use the distance traveled by a fragment in millimeters, and plug that value in for x to find the base pair size for the fragment. The R 2 value shows us that the standard curve is reliable because the value, which is 0.9916, is close to 1, in which 1 represent a perfect graph. Figure 3 shows the distance traveled in millimeters for the DNA Ladder and the digests, as well as the base pair sizes for every one of them. Figure 4 shows the expected fragment sizes for both the single and double digests for my restriction enzymes that I used for pamp, pkan, and pblu, which I calculated using both the New England Biolabs 1 and the DNA Learning Center 2 websites prior to starting this lab. Unfortunately, when I observed Figure 1, I noticed that the double digest did not digest very well, as the band appeared dark in the picture and the fragments were equivalent to the control, confirming that it did not digest. Thus, it was very hard to determine the plasmid by observing the double digest alone. Although the single digest contained some fragments that appeared as it didn t digest, there were a couple of fragments that I was able to analyze and could determine what my plasmid was based off of those fragments. They were the fragments BglI 3 and BglI 5, as shown on Figure 3, with base pair sizes of 4614 and 1092 respectively. When I observed the expected base pair sizes in Figure 4, I found that the base pair sizes for BglI 3 and BglI 5 were closer to pamp for the single digest, with estimated base pair sizes of 3263 and 1118, which were closer to my actual base pair sizes when compared to the estimated sizes for both pkan and pblu as the base pair sizes for each can be seen in Figure 4 for the single digest. Therefore, after analyzing all of this data, I concluded that my plasmid, with a code of 6410F14, is indeed pamp. References Website: 1 New England Biolabs. Internet: < http://tools.neb.com/nebcutter2/> Website:
2 DNA Learning Center. Internet: < http://www.dnalc.org/resources/plasmids.html> Website 3 New England Biolabs. Internet: <https://www.neb.com/tools-and-resources/interactivetools/double-digest-finder>